Liposomal curcumin for treatment of diseases

ABSTRACT

The present invention provides compositions and methods for the treatment of a human patient. The methods and compositions of the present invention include composition for the efficient loading of curcumin, comprising: an amount of a curcuminoid:liposome complex effective to load curcumin into the liposome, wherein the curcuminoids has between 2 to 9 weight percent of the total composition and the curcuminoids are natural or synthetic.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and is a continuation of U.S. patentapplication Ser. No. 11/949,027, filed Dec. 1, 2007, which is acontinuation-in-part of No. U.S. patent application Ser. No. 11/868,251,filed Oct. 5, 2007, which is a continuation-in-part of U.S. patentapplication Ser. No. 11/221,179, filed Sep. 7, 2005, now U.S. Pat. No.7,968,115 issued Jun. 28, 2011, the entire contents of which areincorporated herein by reference.

STATEMENT OF FEDERALLY FUNDED RESEARCH

Not applicable.

FIELD OF THE INVENTION

The invention relates generally to the field of cancer therapeutics, andmore specifically, the invention relates to the use of curcumin orcurcumin analogues to treat a variety of disease conditions.

BACKGROUND OF THE INVENTION

Curcumin (diferuloyl methane) is a natural dietary ingredient, which hasbeen found to have antioxidant and anti-inflammatory properties.Curcumin is found in significant amounts in turmeric, a spice derivedfrom the perennial herb Curcuma longa L. It can suppress the growth ofcertain cancers in the laboratory and prevent the appearance of cancersin animal studies, however the effects of curcumin and curcuminanalogues on cancer cells are highly variable, depending on the type ofcancer studied. The use of curcumin in the treatment ofNeurofibromatosis in vivo, for example, has not, been previouslystudied.

Curcumin and some curcumin derivatives have been previously identifiedas having antioxidant, anti-inflammatory, and in some contexts,antitumor activity when studied in vitro. (Araujo and Leon, 2001).However the antitumor effects are highly unpredictable. For example,Khar et al. found that curcumin induced apoptosis in leukemia, breast,colon, hepatocellular and ovarian carcinoma cell lines in vitro, butfailed to evidence cytotoxic effects in lung, kidney, prostate, cervix,CNS malignancy and melanoma cell lines (Khar et al., 2001). In oneinstance, an in vivo model of human breast cancer showed that curcuminactually inhibited chemotherapy-induced apoptosis of the cancer cellsbeing studied (Somasundaram et al., 2002). The effects of curcumin oncancer cells appear to be variable depending on the specific type ofcancer cell treated.

Oral and topical administration of curcumin has been previously studied.Even at high oral doses, curcumin shows little in the way of toxicity inanimal studies. Studies in rats where the animals were given 1 to 5 g/kgof curcumin found that 75% of the curcumin was excreted in the feces andonly traces appeared in the urine. (Araujo and Leon, 2001). Howeverdespite its low toxicity, curcumin's bioavailability after oraladministration is poor and in vivo concentrations of curcumin that aregrowth inhibitory to tumor cells in vitro cannot be achieved by the oralroute. Intravenous administration of free curcumin has also been foundto be ineffective to achieve significant concentrations of curcumin inany tissue, since curcumin appears to be rapidly metabolized incirculation.

Curcumin has been the subject of several clinical trials in humanpatients, but has only been found to have limited utility in theprevention, and possibly the treatment, of certain cancers of thegastrointestinal tract. Due to the rapid metabolism of curcumin whenadministered orally or intravenously, curcumin therefore has never beenshown to be an effective potential preventative or treatment for cancersother than those of the gastrointestinal tract or cancers where topicalapplication of curcumin would be appropriate. It would therefore bedesirable to identify additional cancers that can be effectively treatedwith curcumin and curcumin analogues, and to develop routes of in vivoadministration of the drug capable of producing concentrations that areinhibitory to tumor cell growth.

Thus, there remains a need in the art for an effective treatment ofdiseases in vivo by curcumin or curcumin analogues. Also, there remainsa need for a more effective means of delivering curcumin or curcuminanalogues to carcinomas than can be provided through oral or topicaldelivery.

SUMMARY OF THE INVENTION

A composition for the efficient loading of curcumin, comprising: anamount of a curcuminoid:liposome complex effective to load curcumin intothe liposome, wherein the curcuminoids comprise between 2 to 9 weightpercent of the total composition and the curcuminoids are natural orsynthetic. In certain aspects, the liposome is PEGylated. In certainaspects, the composition is a DMPC/Chol/DMPE-PEG-2000 liposome at aratio of between 90:10:2 (w/w) to 90:10:9 (w/w) and the curcuminoids; aDMPC/Chol/DSPE-PEG-2000 liposome at a ratio of between 90:10:2 (w/w) to90:10:9 (w/w) and the curcuminoids; a DMPC/DMPE-PEG-2000 liposome at aratio of between 90:10:2 (w/w) to 90:10:9 (w/w) and the curcuminoids; ora DMPC/DSPE-PEG-2000 liposome at a ratio of between 90:10:2 (w/w) to90:10:9 (w/w) and the curcuminoid. The curcuminoid may be administeredin a dose of from about 0.01 mg/kg of the individual's body weight toabout 500 mg/kg of the individual's body weight. The curcumin may beselected from the group consisting of Ar-tumerone, methylcurcumin,demethoxy curcumin, bisdemethoxycurcumin, sodium curcuminate,dibenzoylmethane, acetylcurcumin, feruloyl methane, tetrahydrocurcumin,1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione (curcumin1),1,7-bis(piperonyl)-1,6-heptadiene-3,5-dione (piperonyl curcumin)1,7-bis(2-hydroxy naphthyl)-1,6-heptadiene-2,5-dione (2-hydroxylnaphthyl curcumin) and 1,1-bis(phenyl)-1,3,8,10undecatetraene-5,7-dione.

In one embodiment, the present invention includes a method of treating amalignant or a non-malignant proliferative disease, an autoimmune orauto-inflammatory disease or a degenerative disease comprising providinga patient in need thereof with an effective amount of acurcuminoid:PEGylated-liposome effective to load curcumin into theliposome, wherein the liposome comprises a ratio of liposome to PEGcomprises between 2 to 9 weight percent of the total composition and thecurcuminoids are natural or synthetic and/or the use of a medicamentwith the above characteristics for the treatment of malignant or anon-malignant proliferative disease, an autoimmune or auto-inflammatorydisease or a degenerative disease. In one aspect, the composition usedin the method is a DMPC/Chol/DMPE-PEG-2000 liposome at a ratio ofbetween 90:10:2 (w/w) to 90:10:9 (w/w) and the curcuminoids; aDMPC/Chol/DSPE-PEG-2000 liposome at a ratio of between 90:10:2 (w/w) to90:10:9 (w/w) and the curcuminoids; a DMPC/DMPE-PEG-2000 liposome at aratio of between 90:10:2 (w/w) to 90:10:9 (w/w) and the curcuminoids; ora DMPC/DSPE-PEG-2000 liposome at a ratio of between 90:10:2 (w/w) to90:10:9 (w/w) and the curcuminoid. In one aspect, the curcuminoid isadministered in a dose of from about 0.01 mg/kg of the individual's bodyweight to about 500 mg/kg of the individual's body weight. Examples ofmalignant diseases for treatment using the present invention include,but are not limited to a cancer of the skin, the GI-tract (esophagus,stomach, small and large intestines), the lungs, the liver, thepancreas, the brain, the breasts, the prostate, the uterine cervix andvagina, head and neck and components of the hematopoietic system(leukemias, lymphomas). Non-limiting examples of non-malignant tissueproliferative disease include gastrointestinal polyp formation, multiplepolyposis and neurofibromatosis. Non-limiting examples of autoimmune oranti-inflammatory include anaphylaxis, arthritis, or irritable bowelsyndrome. Examples of neurodegenerative disease include, but are notlimited to, fronto-temporal dementia, Alzheimer's disease, Parkinson'sdisease, Huntington's disease, carpal tunnel syndrome and amyotrophiclateral sclerosis (ALS). Non-limiting examples of degenerative diseasesof the soft-tissue that may be treated using the present inventioninclude cataracts, arthritis, neural disease, muscular disease,connective tissue disease, or a combination thereof. For the method oftreatment and medicaments prepared for use in treated the diseases, thecurcumin may be selected from the group consisting of Ar-tumerone,methylcurcumin, demethoxy curcumin, bisdemethoxycurcumin, sodiumcurcuminate, dibenzoylmethane, acetylcurcumin, feruloyl methane,tetrahydrocurcumin,1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione (curcumin1),1,7-bis(piperonyl)-1,6-heptadiene-3,5-dione (piperonyl curcumin)1,7-bis(2-hydroxy naphthyl)-1,6-heptadiene-2,5-dione (2-hydroxylnaphthyl curcumin) and 1,1-bis(phenyl)-1,3,8,10 undecatetraene-5,7-dioneand combinations thereof.

In yet another embodiment, the present invention includes a method oftreating a parasitic infection by contacting the parasite with aneffective amount of a curcuminoid:liposome complex effective to treatthe parasitic infection and the curcuminoids are natural or syntheticand/or the use of a medicament with the above characteristics for thetreatment of parasitic infections. Non-limiting examples of parasitesthat may be treated using the present invention and a medicamentdirected thereto include falciparum hookworm, filiariais, Leishmaniasis,Treponema, Shistosomaisis. The curcuminoid:liposome complex may alsoinclude one or more anti-malarial agents selected from artesiminin,8-aminoquinoline, amodiaquine, arteether, artemether, artemsinin,artesunate, artesunic acid, artelinic acid, atovoquone, azithromycine,biguanide, chloroquine, chloroquine phosphate, chlorproguanil,cycloguanil, dapsone, desbutyl halofantrine, desipramine, doxycycline,dihydrofolate, reductase inhibitors, dipyridamole, halofantrine,haloperidol, hydroxychloroquine sulfate, imipramine, mefloquine,penfluridol, phospholipid inhibitors, primaquine, proguanil,pyrimethamine, pyronaridine, quinine, quinidine, quinacrineartemisinin,sulfonamides, sulfones, sulfadoxine, sulfalene, tafenoquine,tetracycline, tetrandine, triazine, salts and derivatives thereof.

For the method of treatment and medicaments prepared for use in treatedthe parasitic infections, the curcumin may be selected from the groupconsisting of Ar-tumerone, methylcurcumin, demethoxy curcumin,bisdemethoxycurcumin, sodium curcuminate, dibenzoylmethane,acetylcurcumin, feruloyl methane, tetrahydrocurcumin,1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione (curcumin1),1,7-bis(piperonyl)-1,6-heptadiene-3,5-dione (piperonyl curcumin)1,7-bis(2-hydroxy naphthyl)-1,6-heptadiene-2,5-dione (2-hydroxylnaphthyl curcumin) and 1,1-bis(phenyl)-1,3,8,10 undecatetraene-5,7-dioneand combinations thereof.

Another embodiment of the present invention is a method of treating anon-human animal comprising: providing the non-human animal with aneffective amount of a curcuminoid:liposome complex effective to loadcurcumin into the liposome, wherein the curcuminoids comprises between 2to 9 weight percent of the total composition effective to treat thenon-human animal and the curcuminoids are natural or synthetic and thecurcuminoid:liposome complex is PEGylated. The curcumin may be selectedfrom the group consisting of Ar-tumerone, methylcurcumin, demethoxycurcumin, bisdemethoxycurcumin, sodium curcuminate, dibenzoylmethane,acetylcurcumin, feruloyl methane, tetrahydrocurcumin,1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione (curcumin1),1,7-bis(piperonyl)-1,6-heptadiene-3,5-dione (piperonyl curcumin)1,7-bis(2-hydroxy naphthyl)-1,6-heptadiene-2,5-dione (2-hydroxylnaphthyl curcumin) and 1,1-bis(phenyl)-1,3,8,10 undecatetraene-5,7-dioneand combinations thereof. In one aspect, the curcuminoid:liposomecomplex comprises sterically-stabilized liposomes. Non-limiting examplesof non-human animal include a horse, a cat, a dog, a hamster, a pig, acow, a goat or a non-domesticated animal.

Another embodiment of the present invention is a method of treating ahuman with iron overload or hemochromatosis with a liposomal curcumin orliposomal curcuminoids complex, wherein the complex may be PEGylated ornon-PEGylated and the curcuminoids are natural or synthetic.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1 MTT proliferation/survival assay of Bxpc-3 pancreatic cells.

FIG. 2 MTT proliferation/survival assay of Capan-1 pancreatic cells.

FIG. 3 MTT proliferation/survival assay of Capan-2 pancreatic cells.

FIG. 4 MTT proliferation/survival assay of HS766-T pancreatic cells.

FIG. 5 MTT proliferation/survival assay of ASPC-1 pancreatic cells.

FIG. 6 MTT proliferation/survival assay results for BxPC-3 pancreaticcells.

FIG. 7 MTT proliferation/survival assay results for Capan-1 pancreaticcells.

FIG. 8 MTT proliferation/survival assay results for Capan-2 pancreaticcells.

FIG. 9 MTT proliferation/survival assay results for HS766-T pancreaticcells.

FIG. 10 MTT proliferation/survival assay results for ASPC-1 pancreaticcells.

FIG. 11 Pancreatic BxPC-3 cell recovery of proliferation and survivalafter exposure to liposomal curcumin.

FIG. 12 Pancreatic Capan-1 cell recovery of proliferation and survivalafter exposure to liposomal curcumin.

FIG. 13 Pancreatic Capan-2 cell recovery of proliferation and survivalafter exposure to liposomal curcumin.

FIG. 14 Pancreatic HS766-T cell recovery of proliferation and survivalafter exposure to liposomal curcumin.

FIG. 15 Pancreatic ASPC-1 cell recovery of proliferation and survivalafter exposure to liposomal curcumin.

FIG. 16 Apoptosis assay of the effects of liposomal curcumin onpancreatic BxPC-3 cells.

FIG. 17 Apoptosis assay of the effects of liposomal curcumin onpancreatic HS766-T cells.

FIG. 18 MTT assay of the effects of liposomal curcumin on melanoma A375cells.

FIG. 19 MTT assay of the effects of liposomal curcumin on melanomaHT-144 cells.

FIG. 20 MTT assay of the effects of liposomal curcumin on wild-type,Adriamycin-sensitive MCF-7 breast cancer cells.

FIG. 21 MTT assay of the effects of liposomal curcumin onAdriamycin-resistant MCF-7 breast cancer cells.

FIG. 22 MTT proliferation and survival assay of the effects of liposomalcurcumin on BXPC-3 cells.

FIG. 23 MTT proliferation and survival assay of the effects of liposomalcurcumin on MiaPaCa-2 cells.

FIG. 24 MTT proliferation and survival assay of the effects of liposomalcurcumin on ASPC-1 cells.

FIG. 25 MTT proliferation and survival assay of the effects of liposomalcurcumin on BXPC-3 cells.

FIG. 26 MTT proliferation and survival assay of the effects of liposomalcurcumin on MiaPaCa-2 cells.

DETAILED DESCRIPTION

The present invention provides compositions and methods useful for thetreatment of cancer in which curcumin, or a curcumin analogue havingantitumor activity, is administered parenterally to a patient using acolloidal drug-delivery system. In certain embodiments, the colloidaldrug-delivery system used is a liposomal drug delivery system. In otherembodiments, the colloidal-drug-delivery system used are composed ofmicroparticles (or microspheres), nanoparticles (or nanospheres),nanocapsules, block copolymer micelles, or other polymeric drug deliverysystems. In further embodiments, the drug delivery system used is apolymer-based, non-colloidal drug delivery system such as hydrogels,films or other types of polymeric drug delivery system. In yet furtherembodiments, the curcumin, curcumin analogues or curcumin metabolitesmay be parenterally administered in a lipid-based solvent.

The invention provides compositions and methods useful for treatment orprevention of cancers of any of a wide variety of types, including bothsolid tumors and non-solid tumors such as leukemia and lymphoma. Incertain embodiments, the cancer treated is pancreatic cancer. Thepresent invention can be used to treat either malignant or benigncancers. Carcinomas, sarcomas, myelomas, lymphomas, and leukemias canall be treated using the present invention, including those cancerswhich have a mixed type. Specific types of cancer that can also betreated include, but are not limited to: adenocarcinoma of the breast orprostate; all forms of bronchogenic carcinoma of the lung; myeloid;melanoma; hepatoma; neuroblastoma; papilloma; apudoma; choristoma;branchioma; malignant carcinoid syndrome; carcinoid heart disease;carcinoma (e.g., Walker, basal cell, basosquamous, Brown-Pearce, ductal,Ehrlich tumor, in situ, Krebs 2, merkel cell, mucinous, non-small celllung, oat cell, papillary, scirrhous, bronchiolar, bronchogenic,squamous cell, and transitional cell), histiocytic disorders; leukemia(e.g., B-cell, mixed-cell, null-cell, T-cell, T-cell chronic,HTLV-II-associated, lyphocytic acute, lymphocytic chronic, mast-cell,and myeloid); histiocytosis malignant; Hodgkin's disease;immunoproliferative small; non-Hodgkin's lymphoma; plasmacytoma;reticuloendotheliosis; melanoma; chondroblastoma; chondroma;chondrosarcoma; fibroma; fibrosarcoma; giant cell tumors; histiocytoma;lipoma; liposarcoma; mesothelioma; myxoma; myxosarcoma; osteoma;osteosarcoma; Ewing's sarcoma; synovioma; adenofibroma; adenolymphoma;carcinosarcoma; chordoma; craniopharyngioma; dysgerminoma; hamartoma;mesenchymoma; mesonephroma; myosarcoma; ameloblastoma; cementoma;odontoma; teratoma; thymoma; trophoblastic tumor; adenocarcinoma;adenoma; cholangioma; cholesteatoma; cylindroma; cystadenocarcinoma;cystadenoma; granulosa cell tumor; gynandroblastoma; hepatoma;hidradenoma; islet cell tumor; leydig cell tumor; papilloma; sertolicell tumor; theca cell tumor; leiomyoma; leiomyosarcoma; myoblastoma;myoma; myosarcoma; rhabdomyoma; rhabdomyosarcoma; ependymoma;ganglioneuroma; glioma; medulloblastoma; meningioma; neurilemmoma;neuroblastoma; neuroepithelioma; neurofibroma; neuroma; paraganglioma;paraganglioma nonchromaffin; angiokeratoma; angiolymphoid hyperplasiawith eosinophilia; angioma sclerosing; angiomatosis; glomangioma;hemangioendothelioma; hemangioma; hemangiopericytoma; hemangiosarcoma;lymphangioma; lymphangiomyoma; lymphangiosarcoma; pinealoma;carcinosarcoma; chondrosarcoma; cystosarcoma phyllodes; fibrosarcoma;hemangiosarcoma; leiomyosarcoma; leukosarcoma; liposarcoma;lymphangiosarcoma; myosarcoma; myxosarcoma; ovarian carcinoma;rhabdomyosarcoma; sarcoma (e.g., Ewing's, experimental, Kaposi's, andmast-cell); neoplasms (e.g., bone, breast, digestive system, colorectal,liver, pancreatic, pituitary, testicular, orbital, head and neck,central nervous system, acoustic, pelvic, respiratory tract, andurogenital); neurofibromatosis, and cervical dysplasia), and the like.

The methods of the present invention are useful for the treatment orprevention of cancer in all mammalian subjects, including particularlyhuman patients. As used herein, a patient is a human patient. Also asused herein, treatment means any amelioration of the cancer.

Methods of treatment, as used herein, means the use of compositions ormedicaments to treat patients and other mammalian subjects having cancerin order to at least ameliorate the symptoms of cancer or to halt,inhibit or reverse the progress of the disease. Methods of prevention,as used herein, means to treat patients prophylactically to prevent orinhibit onset of cancer in patients or mammalian subjects who have asusceptibility to developing the disease.

I. Curcumin and Curcumin Analogues.

As used herein curcumin is also known as diferuloylmethane or(E,E)-1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5,-dione andhas the chemical structure depicted below:

Curcumin may be derived from a natural source, the perennial herbCurcuma longa L., which is a member of the Zingiberaceae family. Thespice turmeric is extracted from the rhizomes of Curcuma longa L. andhas long been associated with traditional-medicine treatments used inHindu and Chinese medicine. Turmeric was administered orally ortopically in these traditional treatment methods.

Curcumin is soluble in ethanol, alkalis, ketones, acetic acid andchloroform. It is insoluble in water. Curcumin is therefore lipophilic,and generally readily associates with lipids, e.g. many of those used inthe colloidal drug-delivery systems of the present invention. In certainembodiments, curcumin can also be formulated as a metal chelate.

As used herein, curcumin analogues are those compounds which due totheir structural similarity to curcumin, exhibit anti-proliferative orpro-apoptotic effects on cancer cells similar to that of curcumin.Curcumin analogues which may have anti-cancer effects similar tocurcumin include Ar-tumerone, methylcurcumin, demethoxy curcumin,bisdemethoxycurcumin, sodium curcuminate, dibenzoylmethane,acetylcurcumin, feruloyl methane, tetrahydrocurcumin,1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione (curcumin1),1,7-bis(piperonyl)-1,6-heptadiene-3,5-dione (piperonyl curcumin)1,7-bis(2-hydroxy naphthyl)-1,6-heptadiene-2,5-dione (2-hydroxylnaphthyl curcumin), 1,1-bis(phenyl)-1,3,8,10 undecatetraene-5,7-dione(cinnamyl curcumin) and the like (Araujo and Leon, 2001; Lin et al.,2001; John et al., 2002; see also Ishida et al., 2002). Curcuminanalogues may also include isomers of curcumin, such as the (Z,E) and(Z,Z) isomers of curcumin. In a related embodiment, curcumin metaboliteswhich have anti-cancer effects similar to curcumin can also be used inthe present invention. Known curcumin metabolites include glucoronidesof tetrahydrocurcumin and hexahydrocurcumin, and dihydroferulic acid. Incertain embodiments, curcumin analogues or metabolites can be formulatedas metal chelates, especially copper chelates. Other appropriatederivatives of curcumin, curcumin analogues and curcumin metabolitesappropriate for use in the present invention will be apparent to one ofskill in the art.

As used herein, “active ingredient” refers to curcumin, or a curcuminanalogue or metabolite which exhibits anti-cancer activity whenadministered to a cancer patient.

II. Liposomes and Other Colloidal Drug Delivery Vehicles.

Colloidal drug delivery vehicles including liposomes can be used in thepresent invention to deliver curcumin or curcumin analogues ormetabolites to cancer cells in a patient. Curcumin or a curcuminanalogue or metabolite is encapsulated in a colloidal drug deliveryvehicle that is capable of delivering the drug to target cancer cells.Whenever “encapsulation” is used in this patent it is meant to includeincorporation as well.

As used herein, a colloidal drug delivery vehicle is one that comprisesparticles that are capable of being suspended in a pharmaceuticallyacceptable liquid medium wherein the size range of the particles rangesfrom several nanometers to several micrometers in diameter. Thecolloidal drug delivery systems contemplated by the present inventionparticularly include those that substantially retain their colloidalnature when administered in vivo. Colloidal drug delivery systemsinclude, but are not limited to, lipid-based and polymer-basedparticles. Examples of colloidal drug delivery systems includeliposomes, nanoparticles, (or nanospheres), nanocapsules, microparticles(or microspheres), and block copolymer micelles.

Liposomes bear many resemblances to cellular membranes and arecontemplated for use in connection with the present invention ascarriers for curcumin, curcumin analogues, curcumin metabolites, orderivatives of curcumin, curcumin analogues or curcumin metabolites.They are widely suitable as both water- and lipid-soluble substances canbe encapsulated, i.e., in the aqueous spaces and within the bilayeritself, respectively. The liposomal formulation of the liposome can bemodified by those of skill in the art to maximize the solubility ofcurcumin or a curcumin analogue or metabolite based on theirhydrophobicity. Curcumin, for example, is a water insoluble compoundthat is soluble in ethanol, ketone, and chloroform and therefore wouldbe expected to be relatively lipophilic. It is also possible to employliposomes for site-specific delivery of the active ingredient byselectively modifying the liposomal formulation.

III. Lipid Composition of Liposomes.

Liposomes suitable for use in the delivery of curcumin or curcuminanalogues or metabolites include those composed primarily ofvesicle-forming lipids. Appropriate vesicle-forming lipids for use inthe present invention include those lipids which can form spontaneouslyinto bilayer vesicles in water, as exemplified by the phospholipids.

Selection of the appropriate lipids for liposome composition is governedby the factors of: (1) liposome stability, (2) phase transitiontemperature, (3) charge, (4) non-toxicity to mammalian systems, (5)encapsulation efficiency, (6) lipid mixture characteristics. It isexpected that one of skill in the art who has the benefit of thisdisclosure could formulate liposomes according to the present inventionwhich would optimize these factors. The vesicle-forming lipids of thistype are preferably ones having two hydrocarbon chains, typically acylchains, and a head group, either polar or nonpolar. The hydrocarbonchains may be saturated or have varying degrees of unsaturation. Thereare a variety of synthetic vesicle-forming lipids andnaturally-occurring vesicle-forming lipids, including the sphingolipids,ether lipids, sterols, phospholipids, particularly thephosphoglycerides, and the glycolipids, such as the cerebrosides andgangliosides.

Phosphoglycerides include phospholipids such as phosphatidylcholine,phosphatidylethanolamine, phosphatidic acid, phosphatidylinositol,phosphatidylserine phosphatidylglycerol and diphosphatidylglycerol(cardiolipin), where the two hydrocarbon chains are typically betweenabout 14-22 carbon atoms in length, and have varying degrees ofunsaturation. As used herein, the abbreviation “PC” stands forphosphatidylcholine, and “PS” stand for phosphatidylserine. Lipidscontaining either saturated and unsaturated fatty acids are widelyavailable to those of skill in the art. Additionally, the twohydrocarbon chains of the lipid may be symmetrical or asymmetrical. Theabove-described lipids and phospholipids whose acyl chains have varyinglengths and degrees of saturation can be obtained commercially orprepared according to published methods.

Exemplary phosphatidylcholines include dilauroyl phophatidylcholine,dimyristoylphophatidylcholine, dipalmitoylphophatidylcholine,distearoylphophatidyl-choline, diarachidoylphophatidylcholine,dioleoylphophatidylcholine, dilinoleoyl-phophatidylcholine,dierucoylphophatidylcholine, palmitoyl-oleoyl-phophatidylcholine, eggphosphatidylcholine, myristoyl-palmitoylphosphatidylcholine,palmitoyl-myristoyl-phosphatidylcholine,myristoyl-stearoylphosphatidylcholine,palmitoyl-stearoylphosphatidylcholine,stearoyl-palmitoylphosphatidylcholine,stearoyl-oleoyl-phosphatidylcholine,stearoyl-linoleoylphosphatidylcholine andpalmitoyl-linoleoylphosphatidylcholine. Assymetric phosphatidylcholinesare referred to as 1-acyl, 2-acyl-sn-glycero-3-phosphocholines, whereinthe acyl groups are different from each other. Symmetricphosphatidylcholines are referred to as1,2-diacyl-sn-glycero-3-phosphocholines. As used herein, theabbreviation “PC” refers to phosphatidylcholine. The phosphatidylcholine1,2-dimyristoyl-sn-glycero-3-phosphocholine is abbreviated herein as“DMPC.” The phosphatidylcholine 1,2-dioleoyl-sn-glycero-3-phosphocholineis abbreviated herein as “DOPC.” The phosphatidylcholine1,2-dipalmitoyl-sn-glycero-3-phosphocholine is abbreviated herein as“DPPC.”

In general, saturated acyl groups found in various lipids include groupshaving the trivial names propionyl, butanoyl, pentanoyl, caproyl,heptanoyl, capryloyl, nonanoyl, capryl, undecanoyl, lauroyl,tridecanoyl, myristoyl, pentadecanoyl, palmitoyl, phytanoyl,heptadecanoyl, stearoyl, nonadecanoyl, arachidoyl, heniecosanoyl,behenoyl, trucisanoyl and lignoceroyl. The corresponding IUPAC names forsaturated acyl groups are trianoic, tetranoic, pentanoic, hexanoic,heptanoic, octanoic, nonanoic, decanoic, undecanoic, dodecanoic,tridecanoic, tetradecanoic, pentadecanoic, hexadecanoic,3,7,11,15-tetramethylhexadecanoic, heptadecanoic, octadecanoic,nonadecanoic, eicosanoic, heneicosanoic, docosanoic, trocosanoic andtetracosanoic. Unsaturated acyl groups found in both symmetric andassymmetric phosphatidylcholines include myristoleoyl, palmitoleoyl,oleoyl, elaidoyl, linoleoyl, linolenoyl, eicosenoyl and arachidonoyl.The corresponding IUPAC names for unsaturated acyl groups are9-cis-tetradecanoic, 9-cis-hexadecanoic, 9-cis-octadecanoic,9-trans-octadecanoic, 9-cis-12-cis-octadecadienoic,9-cis-12-cis-15-cis-octadecatrienoic, 11-cis-eicosenoic and5-cis-8-cis-11-cis-14-cis-eicosatetraenoic.

Alternately, U.S. Pat. No. 5,972,380 describes the use of “caged”phospholipids. Caged phospholipids are aminophospholipids that whenpresent in a liposome, render the liposome pH-sensitive so that onceendocytosed into target cells the caging groups are cleaved. Thiscleavage results in destabilization of the liposome, causing the uncagedlipids of the liposome to become fusogenic and to thereby release of theactive agent carried by the liposome into the cell cytosome.

Exemplary phosphatidylethanolamines includedimyristoyl-phosphatidylethanolamine,dipalmitoyl-phosphatidylethanolamine,distearoyl-phosphatidylethanolamine, dioleoyl-phosphatidylethanolamineand egg phosphatidylethanolamine. Phosphatidylethanolamines may also bereferred to under IUPAC naming systems as1,2-diacyl-sn-glycero-3-phosphoethanolamines or1-acyl-2-acyl-sn-glycero-3-phosphoethanolamine, depending on whetherthey are symmetric or assymetric lipids.

Exemplary phosphatidic acids include dimyristoyl phosphatidic acid,dipalmitoyl phosphatidic acid and dioleoyl phosphatidic acid.Phosphatidic acids may also be referred to under IUPAC naming systems as1,2-diacyl-sn-glycero-3-phosphate or1-acyl-2-acyl-sn-glycero-3-phosphate, depending on whether they aresymmetric or assymetric lipids.

Exemplary phosphatidylserines include dimyristoyl phosphatidylserine,dipalmitoyl phosphatidylserine, dioleoylphosphatidylserine, distearoylphosphatidylserine, palmitoyl-oleylphosphatidylserine and brainphosphatidylserine. Phosphatidylserines may also be referred to underIUPAC naming systems as 1,2-diacyl-sn-glycero-3-[phospho-L-serine] or1-acyl-2-acyl-sn-glycero-3-[phospho-L-serine], depending on whether theyare symmetric or assymetric lipids. As used herein, the abbreviation“PS” refers to phosphatidylserine.

Exemplary phosphatidylglycerols include dilauryloylphosphatidylglycerol,dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol,dioleoyl-phosphatidylglycerol, dimyristoylphosphatidylglycerol,palmitoyl-oleoyl-phosphatidylglycerol and egg phosphatidylglycerol.Phosphatidylglycerols may also be referred to under IUPAC naming systemsas 1,2-diacyl-sn-glycero-3-[phospho-rac-(1-glycerol)] or1-acyl-2-acyl-sn-glycero-3-[phospho-rac-(1-glycerol)], depending onwhether they are symmetric or assymetric lipids. Thephosphatidylglycerol1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] is abbreviatedherein as “DMPG”. The phosphatidylglycerol1,2-dipalmitoyl-sn-glycero-3-(phospho-rac-1-glycerol) (sodium salt) isabbreviated herein as “DPPG”.

Suitable sphingomyelins might include brain sphingomyelin, eggsphingomyelin, dipalmitoyl sphingomyelin, and distearoyl sphingomyelin.

Other suitable lipids include glycolipids, sphingolipids, ether lipids,glycolipids such as the cerebrosides and gangliosides, and sterols, suchas cholesterol or ergosterol. As used herein, the term cholesterol issometimes abbreviated as “Chol.”

Additional lipids suitable for use in liposomes are known to persons ofskill in the art and are cited in a variety of sources, such as 1998McCutcheon's Detergents and Emulsifiers, 1998 McCutcheon's FunctionalMaterials, both published by McCutcheon Publishing Co., New Jersey, andthe Avanti Polar Lipids, Inc. Catalog.

Suitable lipids for use in the present invention will have sufficientlong-term stability to achieve an adequate shelf-life. Factors affectinglipid stability are well-known to those of skill in the art and includefactors such as the source (e.g. synthetic or tissue-derived), degree ofsaturation and method of storage of the lipid.

The overall surface charge of the liposome can affect the tissue uptakeof a liposome. In certain embodiments of the present invention anionicphospholipids such as phosphatidylserine, phosphatidylinositol,phosphatidic acid, and cardiolipin will be suitable for use in thepresent invention. Also, neutral lipids such as dioleoylphosphatidylethanolamine (DOPE) may be used to target uptake of liposomes byspecific tissues or to increase circulation times of intravenouslyadministered liposomes. Further, cationic lipids may be used in thepresent invention for alteration of liposomal charge, where the cationiclipid can be included as a minor component of the lipid composition oras a major or sole component. Suitable cationic lipids typically have alipophilic moiety, such as a sterol, an acyl or diacyl chain, and wherethe lipid has an overall net positive charge. Preferably, the head groupof the lipid carries the positive charge.

One of skill in the art will select vesicle-forming lipid that achieve aspecified degree of fluidity or rigidity. The fluidity or rigidity ofthe liposome can be used to control factors such as the stability of theliposome in serum or the rate of release of the entrapped agent in theliposome.

Liposomes having a more rigid lipid bilayer, or a liquid crystallinebilayer, are achieved by incorporation of a relatively rigid lipid. Therigidity of the lipid bilayer correlates with the phase transitiontemperature of the lipids present in the bilayer. Phase transitiontemperature is the temperature at which the lipid changes physical stateand shifts from an ordered gel phase to a disordered liquid crystallinephase. Several factors affect the phase transition temperature of alipid including hydrocarbon chain length and degree of unsaturation,charge and headgroup species of the lipid. Lipid having a relativelyhigh phase transition temperature will produce a more rigid bilayer.Other lipid components, such as cholesterol, are also known tocontribute to membrane rigidity in lipid bilayer structures. Cholesterolis widely used by those of skill in the art to manipulate the fluidity,elasticity and permeability of the lipid bilayer. It is thought tofunction by filling in gaps in the lipid bilayer. In contrast, lipidfluidity is achieved by incorporation of a relatively fluid lipid,typically one having a lower phase transition temperature. Phasetransition temperatures of many lipids are tabulated in a variety ofsources, such as Avanti Polar Lipids catalogue and Lipidat by MartinCaffrey, CRC Press.

Non-toxicity of the lipids is also a significant consideration in thepresent invention. Lipids approved for use in clinical applications arewell-known to those of skill in the art. In certain embodiments of thepresent invention, synthetic lipids, for example, may be preferred overlipids derived from biological sources due to a decreased risk of viralor protein contamination from the source organism.

IV. Liposome Formation and Curcumin Entrapment.

The formation and use of liposomes is generally known to those of skillin the art, as described in, e.g. Liposome Technology, Vols. 1, 2 and 3,Gregory Gregoriadis, ed., CRC Press, Inc; Liposomes: Rational Design,Andrew S. Janoff, ed., Marcel Dekker, Inc.; Medical Applications ofLiposomes, D. D. Lasic and D. Papahadjopoulos, eds., Elsevier Press;Bioconjugate Techniques, by Greg T. Hermanson, Academic Press; andPharmaceutical Manufacturing of Liposomes, by Francis J. Martin, inSpecialized Drug Delivery Systems (Praveen Tyle, Ed.), Marcel Dekker,Inc.

The original method of forming liposomes (Bangham et al., 1965, J. Mol.Biol. 13: 238-252) involved first suspending phospholipids in an organicsolvent and then evaporating to dryness until a dry lipid cake or filmis formed. An appropriate amount of aqueous medium is added and thelipids spontaneously form multilamellar concentric bilayer vesicles(also termed multilamellar vesicles (MLVs). These MLVs can then bedispersed by mechanical means. MLVs generally have diameters of from 25nm to 4 μm. Sonication of MLVs results in the formation of smallunilamellar vesicles (SUVs) with diameters in the range of 200 to 500 Å,containing an aqueous solution in the core. SUVs are smaller than MLVsand unilamellar.

While the original MLVs and SUVs were created using phospholipids, anyof the lipid compositions described previously can be used to createMLVs and SUVs. When mixtures of lipids are used the lipids are typicallyco-dissolved in an organic solvent prior to the evaporation step of theprocess described above.

An alternate method of creating large unilamellar vesicles (LUVs) is thereverse-phase evaporation process, described, for example, in U.S. Pat.No. 4,235,871. This process generates reverse-phase evaporation vesicles(REVs), which are mostly unilamellar but also typically contain someoligolamellar vesicles. In this procedure a mixture of polar lipid in anorganic solvent is mixed with a suitable aqueous medium. A homogeneouswater-in-oil type of emulsion is formed and the organic solvent isevaporated until a gel is formed. The gel is then converted to asuspension by dispersing the gel-like mixture in an aqueous media.

Liposomes of the present invention may also be prepared wherein theliposomes have substantially homogeneous sizes in a selected size range.One effective sizing method for REVs and MLVs involves extruding anaqueous suspension of the liposomes through a series of polycarbonatemembranes having a selected uniform pore size in the range of 0.03 to0.2 micron, typically 0.05, 0.08, 0.1, or 0.2 microns. The pore size ofthe membrane corresponds roughly to the largest sizes of liposomesproduced by extrusion through that membrane, particularly where thepreparation is extruded two or more times through the same membrane.Homogenization methods are also useful for down-sizing liposomes tosizes of 100 nm or less (Martin, F. J., in Specialized Drug DeliverySystems-Manufacturing and Production Technology, (P. Tyle, Ed.) MarcelDekker, New York, pp. 267-316 (1990)). Homogenization relies on shearingenergy to fragment large liposomes into smaller ones. Other appropriatemethods of down-sizing liposomes include reducing liposome size byvigorous agitation of the liposomes in the presence of an appropriatesolubilizing detergent, such as deoxycholate.

Liposomes that have been sized to a range of about 0.2-0.4 microns maybe sterilized by filtering the liposomes through a conventionalsterilization filter, which is typically a 0.22 micron filter, on a highthroughput basis. Other appropriate methods of sterilization will beapparent to those of skill in the art.

In certain embodiments, curcumin or curcumin analogues or metabolitescan be incorporated into liposomes by several standard methods. Becausecurcumin is water-insoluble, it is possible to passively entrap curcuminor lipophilic curcumin analogues or metabolites by hydrating a lipidfilm or lipid emulsion that already contains an appropriateconcentration of curcumin or a curcumin analogue. Alternately, curcuminor a curcumin analogue could be passively entrapped within the liposomeby generating liposomes in a suitable polar solvent, such as ethanol, inwhich an appropriate concentration of curcumin or a curcumin analog hasbeen dissolved, and subsequently dispersing the liposomes in an aqueousmedium. Water-soluble curcumin analogues or metabolites could bepassively entrapped by hydrating a lipid film with an aqueous solutionof the agent.

In an alternate embodiment of the present invention, curcumin orcurcumin analogues or metabolites can be conjugated to the surface ofthe liposomal bilayer. One well-known method of covalently attaching adrug to a liposome is the use of amide conjugation. For example,phospholipids having amine functional groups can be conjugated to one ofthe hydroxyl groups found in curcumin and various curcumin analogues ormetabolites. Suitable lipids for amide conjugation to curcumin mightinclude phosphatidylethanolamines andN-caproylamine-phosphatidylethanolamines. Curcumin analogues ormetabolites containing other suitable functional groups, such ascarboxyl groups or amine groups, can also be used in amide conjugationto a vesicle-forming lipid having a suitable functional group.

Phospholipids can form a variety of structures other than liposomes whendispersed in water, depending on the molar ratio of lipid to water. Atlow ratios the liposome is the preferred structure. The physicalcharacteristics of liposomes depend on the pH, ionic strength and thepresence of divalent cations. Liposomes can show low permeability toionic and polar substances, but at elevated temperatures undergo a phasetransition which markedly alters their permeability. The phasetransition involves a change from a closely packed, ordered structure,known as the gel state, to a loosely packed, less-ordered structure,known as the fluid state. This occurs at a characteristicphase-transition temperature and results in an increase in permeabilityto ions, sugars and drugs.

In addition to temperature, exposure to proteins can alter thepermeability of liposomes. Certain soluble proteins such as cytochrome cbind, deform and penetrate the bilayer, thereby causing changes inpermeability. Cholesterol inhibits this penetration of proteins,apparently by packing the phospholipids more tightly.

The ability to trap solutes varies between different types of liposomes.For example, MLVs are moderately efficient at trapping solutes, but SUVsare extremely inefficient. SUVs offer the advantage of homogeneity andreproducibility in size distribution, however, and a compromise betweensize and trapping efficiency is offered by large unilamellar vesicles(LUVs). These are prepared by ether evaporation and are three to fourtimes more efficient at solute entrapment than MLVs.

In addition to liposome characteristics, an important determinant inentrapping compounds is the physicochemical properties of the compounditself. Polar compounds are trapped in the aqueous spaces and nonpolarcompounds bind to the lipid bilayer of the vesicle. Polar compounds arereleased through permeation or when the bilayer is broken, but nonpolarcompounds remain affiliated with the bilayer unless it is disrupted bytemperature or exposure to lipoproteins. Both types show maximum effluxrates at the phase transition temperature.

V. Liposome Targeting Techniques.

Liposomes interact with cells via four different mechanisms: (1)endocytosis by phagocytic cells of the reticuloendothelial system suchas macrophages and neutrophils; (2) adsorption to the cell surface,either by nonspecific weak hydrophobic or electrostatic forces, or byspecific interactions with cell-surface components; (3) fusion with theplasma cell membrane by insertion of the lipid bilayer of the liposomeinto the plasma membrane, with simultaneous release of liposomalcontents into the cytoplasm; and (4) by transfer of liposomal lipids tocellular or subcellular membranes, or vice versa, without anyassociation of the liposome contents. It often is difficult to determinewhich mechanism is operative and more than one may operate at the sametime.

The fate and disposition of intravenously injected liposomes depend ontheir physical properties, such as size, fluidity and surface charge.They may persist in tissues for hours or days, depending on theircomposition, and half lives in the blood range from minutes to severalhours. Larger liposomes, such as MLVs and LUVs, are taken up rapidly byphagocytic cells of the reticuloendothelial system, but physiology ofthe circulatory system restrains the exit of such large species at mostsites. They can exit only in places where large openings or pores existin the capillary endothelium, such as the sinusoids of the liver orspleen. Thus, these organs are the predominate site of uptake. On theother hand, SUVs show a broader tissue distribution but still aresequestered highly in the liver and spleen. In general, this in vivobehavior limits the potential targeting of liposomes to only thoseorgans and tissues accessible to their large size. These include theblood, liver, spleen, bone marrow and lymphoid organs.

Smaller liposomes may exit the circulatory system at points where theendothelium has become “leaky”. Solid tumors and inflammation sitesoften produce leaky endothelium that permits the extravasion of smallliposomes. This effect is greatly enhanced by increasing the circulationtimes of the liposomes so that the liposomes may take advantage of thiseffect. One way of increasing the circulation time of liposomes is byusing STEALTH® liposomes. STEALTH® liposomes are typically derivatizedwith a hydrophilic polymer chain or polyalkylether, such aspolyethyleneglycol (PEG). (See, for example, U.S. Pat. Nos. 5,013,556,5,213,804, 5,225,212 and 5,395,619, herein incorporated by reference.)The polymer coating reduces the rate of uptake of liposomes bymacrophages and thereby prolongs the presence of the liposomes in theblood stream. This can also be used as a mechanism of prolonged releasefor the drugs carried by the liposomes, (see e.g. Woodle et al., 1992).In the present invention, therefore, it will be desirable in certainembodiments that liposomal curcumin be delivered by aSTEALTH®-liposome-type derivatized liposome formulation such asPEGylated liposomes. PEGylated liposomes are also referred to herein assterically-stabilized liposomes or “SSL,” in contrast to non-PEGylatedliposomes which are referred to as conventional liposomes or “CL.”

Polyethylene glycol-lipid conjugates can be produced using a variety oflipids. Pegylated lipids that are currently commercially availableinclude the phosphatidylethanolamines1,2-diacyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethyleneglycol)-550], 1,2-diacyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-750],1,2-diacyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethyleneglycol)-1000], 1,2-diacyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000],1,2-diacyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethyleneglycol)-3000] and 1,2-diacyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000]. As used herein, “DMPE-PEG-2000” stands for1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt). As used herein,“DSPE-PEG-2000” stands for1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethyleneglycol)-2000] (ammonium salt).

In other embodiments, it may further be desirable to target curcumindelivery to specific tissues, particularly tumor tissues. Shouldspecific targeting be desired, methods are available for this to beaccomplished. Targeting ligands such as antibodies or antibody fragmentscan be used to bind to the liposome surface and to direct the antibodyand its drug contents to specific antigenic receptors located on aparticular cell-type surface. (See e.g., Mastrobattista et al., 1999)Carbohydrate determinants (glycoprotein or glycolipid cell-surfacecomponents that play a role in cell-cell recognition, interaction andadhesion) can also be used as targeting ligands as they have potentialin directing liposomes to particular cell types. Certain proteins can beused as targeting ligands, usually ones that are recognized byself-surface receptors of the targeted tissue. For example, a ligandthat binds to a cell-surface receptor that is overexpressed inparticular cancer cells might be used to increase uptake of liposomes bythe target tissue. Cell surface receptors that are endocytosed will bepreferred in certain embodiments. When combined with “stealth”technology, the targeting ligand is often attached to the end of thehydrophilic polymer that is exposed to the aqueous medium. Alternately,liposomes can incorporate fusogenic proteins, e.g. fusogenic proteinsderived from viruses, which induce fusion of the liposome with thecellular membrane.

As used herein, targeting ligands are any ligand which causes liposomesto associate with the target cell-type to an enhanced degree overnon-targeted tissues. In certain embodiments, the targeting ligand is acell surface receptor that is endocytosed by the target cell.Appropriate targeting ligands for use in the present invention includeany ligand that causes increased binding or association of liposomeswith cell-surface of the target cells over non-target cells, includingantibodies, antibody fragments, carbohydrate determinants, or proteins,preferably proteins that are recognized by cell-surface receptors orfusogenic proteins.

Passive targeting of liposomes relies on non-specific release of thedrug payload over time, and does not rely on the use of targetingligands. Targeting-ligand containing liposomes however can achievecytosolic delivery of curcumin or curcumin analogues or metabolites by anumber of additional mechanisms. Liposomes can be actively targeted to aparticular cell type, and release the contents of the liposomeextracellularly in the vicinity of the target tissue, or transferlipophilic compounds directly from the liposome to the cell membrane.Alternately, the target-ligand containing liposome can bind to a cellsurface receptor that is endocytosed, which would permit intracellularrelease of the liposomal drug payload. Fusogenic peptides orpH-sensitive liposomes are particularly useful in this context totrigger release of the liposomal contents from the endosome into thecytoplasm.

VI. Polymeric Drug Delivery Systems.

The present invention also provides that curcumin or curcumin analoguecan be encapsulated within a protective wall material that is polymericin nature rather than lipid-based. The polymer used to encapsulate thebioactive agent is typically a single copolymer or homopolymer. Thepolymeric drug delivery system may be colloidal or non-colloidal innature.

Colloidal polymeric encapsulation structures include microparticles,microcapsules, microspheres, nanoparticles, nanocapsules andnanospheres, block copolymer micelles, or any other encapsulatedstructure. Both synthetic polymers, which are made by man, andbiopolymers, including proteins and polysaccharides, can be used in thepresent invention. The polymeric drug delivery system may be composed ofbiodegradable or non-biodegradable polymeric materials, or anycombination thereof.

As used herein, the term “microparticles” (or “microspheres”) refers toa solid object, essentially of regular or semi-regular shape, that ismore than about one micrometer in its largest diameter and exhibits aliquid core and semipermeable polymeric shell. Microparticles ormicrospheres typically have a diameter of from about 1 to 2,000 μm (2mm), normally ranging from about 100 to 500 μm.

As used herein, the term “nanoparticle” (or “nanosphere”) refers to asubmicroscopic solid object, essentially of regular or semi-regularshape, that is less than one micrometer in its largest dimension andexhibits a liquid core and a semipermeable polymeric shell.Nanoparticles or nanospheres typically range from about 1 to 1,000nanometers (nm). Normally, nanoparticles range from about 100 to 300 nm.

As used herein, the term “microcapsule” refers to a microscopic (fewmicrometers in size to few millimeters) solid object of from a fewmicrometers to a few millimeters in size that is of essentially regularcylindrical shape and exhibits a liquid core and a semipermeablepolymeric shell.

As used herein, the term “nanocapsules” refers to a solid object of lessthan about one micrometer in size that is essentially regular in shapeand which exhibits a liquid core and a semipermeable polymeric shell.

Block copolymer micelles are formed from two or more monomeric unitswhich, following polymerization, are arranged in a specific mannerdepending on the type of copolymer desired. Micelles are formed fromindividual block copolymer molecules, each of which contains ahydrophobic block and a hydrophilic block. The amphiphilic nature of theblock copolymers enables them to self-assemble to form nanosizedaggregates of various morphologies in aqueous solution such that thehydrophobic blocks form the core of the micelle, which is surrounded bythe hydrophilic blocks, which form the outer shell (Zhang L. EisenbergA. (1995) Science, 268:1728-1731; Zhang L, Yu K., Eisenberg A. (1996)Science, 272:1777-1779). The inner core of the micelle creates ahydrophobic microenvironment for the non-polar drug, while thehydrophilic shell provides a stabilizing interface between the micellecore and the aqueous medium. The properties of the hydrophilic shell canbe adjusted to both maximize biocompatibility and avoidreticuloendothelial system uptake and renal filtration. The size of themicelles is usually between 10 nm and 100 nm.

Non-colloidal polymeric drug-delivery systems including films, hydrogelsand “depot” type drug delivery systems are also contemplated by thepresent invention. Such non-colloidal polymeric systems can also be usedin the present invention in conjunction with parenteral injection,particularly where the non-colloidal drug delivery system is placed inproximity to the targeted cancerous tissue.

As used herein, the term “hydrogel” refers to a solution of polymers,sometimes referred to as a sol, converted into gel state by small ionsor polymers of the opposite charge or by chemical crosslinking.

As used herein, the term “polymeric film” refers to a polymer-based filmgenerally from about 0.5 to 5 mm in thickness which is sometimes used asa coating.

In certain embodiments the microparticles, nanoparticles, microcapsules,block copolymer micelles or other polymeric drug delivery systemscomprising curcumin or a curcumin analogue can be coupled with atargeting or binding partner. By linking the polymeric drug deliverysystem to one or more binding proteins or peptides, delivery of theencapsulated therapeutic agent can be directed to a target cellpopulation which binds to the binding protein or peptide.

VII. Pharmaceutical Compositions.

As used herein, the term ‘pharmaceutically acceptable’ (or‘pharmacologically acceptable’) refers to molecular entities andcompositions that do not produce an adverse, allergic or other untowardreaction when administered to an animal or a human, as appropriate. Theterm ‘pharmaceutically acceptable carrier’, as used herein, includes anyand all solvents, dispersion media, coatings, antibacterial, isotonicand absorption delaying agents, buffers, excipients, binders,lubricants, gels, surfactants and the like, that may be used as a mediafor a pharmaceutically acceptable substance.

Pharmaceutical compositions of the present invention comprising curcuminor a curcumin analogue and a colloidal drug delivery carrier such as aliposome are prepared according to standard techniques. (As used herein,the abbreviation “L-cur” refers to liposomal curcumin compositions.)They can further comprise a pharmaceutically acceptable carrier.Generally, a pharmaceutical carrier such as normal saline will beemployed. Other suitable carriers include water, buffered water,isotonic aqueous solutions, 0.4% saline, 0.3% aqueous glycine and thelike, including glycoproteins for enhanced stability, such as albumin,lipoprotein and globulin. These compositions can be sterilized byconventional sterilization techniques that are well-known to those ofskill in the art. Sufficiently small liposomes, for example, can besterilized using sterile filtration techniques.

Formulation characteristics that can be modified include, for example,the pH and the osmolality. For example, it may be desired to achieve aformulation that has a pH and osmolality similar to that of human bloodor tissues to facilitate the formulation's effectiveness whenadministered parenterally. Alternatively, to promote the effectivenessof the disclosed compositions when administered via other administrationroutes, alternative characteristics may be modified.

Buffers are useful in the present invention for, among other purposes,manipulation of the total pH of the pharmaceutical formulation(especially desired for parenteral administration). A variety of buffersknown in the art can be used in the present formulations, such asvarious salts of organic or inorganic acids, bases, or amino acids, andincluding various forms of citrate, phosphate, tartrate, succinate,adipate, maleate, lactate, acetate, bicarbonate, or carbonate ions.Particularly advantageous buffers for use in parenterally administeredforms of the presently disclosed compositions in the present inventioninclude sodium or potassium buffers, particularly sodium phosphate. In apreferred embodiment for parenteral dosing, sodium phosphate is employedin a concentration approximating 20 mM to achieve a pH of approximately7.0. A particularly effective sodium phosphate buffering systemcomprises sodium phosphate monobasic monohydrate and sodium phosphatedibasic heptahydrate. When this combination of monobasic and dibasicsodium phosphate is used, advantageous concentrations of each are about0.5 to about 1.5 mg/ml monobasic and about 2.0 to about 4.0 mg/mldibasic, with preferred concentrations of about 0.9 mg/ml monobasic andabout 3.4 mg/ml dibasic phosphate. The pH of the formulation changesaccording to the amount of buffer used.

Depending upon the dosage form and intended route of administration itmay alternatively be advantageous to use buffers in differentconcentrations or to use other additives to adjust the pH of thecomposition to encompass other ranges. Useful pH ranges for compositionsof the present invention include a pH of about 2.0 to a pH of about12.0.

In some embodiments, it will also be advantageous to employ surfactantsin the presently disclosed formulations, where those surfactants willnot be disruptive of the drug-delivery system used. Surfactants oranti-adsorbants that prove useful include polyoxyethylenesorbitans,polyoxyethylenesorbitan monolaurate, polysorbate-20, such as Tween-20™,polysorbate-80, hydroxycellulose, and genapol. By way of example, whenany surfactant is employed in the present invention to produce aparenterally administrable composition, it is advantageous to use it ina concentration of about 0.01 to about 0.5 mg/ml.

Additional useful additives are readily determined by those of skill inthe art, according to particular needs or intended uses of thecompositions and formulator. One such particularly useful additionalsubstance is sodium chloride, which is useful for adjusting theosmolality of the formulations to achieve the desired resultingosmolality. Particularly preferred osmolalities for parenteraladministration of the disclosed compositions are in the range of about270 to about 330 mOsm/kg. The optimal osmolality for parenterallyadministered compositions, particularly injectables, is approximately300 Osm/kg and achievable by the use of sodium chloride inconcentrations of about 6.5 to about 7.5 mg/ml with a sodium chlorideconcentration of about 7.0 mg/ml being particularly effective.

Curcumin-containing liposomes or curcumin-containing colloidaldrug-delivery vehicles can be stored as a lyophilized powder underaseptic conditions and combined with a sterile aqueous solution prior toadministration. The aqueous solution used to resuspend the liposomes cancontain pharmaceutically acceptable auxiliary substances as required toapproximate physical conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents and the like, as discussed above.

In other embodiments the curcumin-containing liposomes orcurcumin-containing colloidal drug-delivery vehicle can be stored as asuspension, preferable an aqueous suspension, prior to administration.In certain embodiments, the solution used for storage of liposomes orcolloidal drug carrier suspensions will include lipid-protective agentswhich protect lipids against free-radical and lipid-peroxidative damageon storage. Suitable protective compounds include free-radical quencherssuch as alpha-tocopherol and water-soluble iron-specific chelators, suchas ferrioxamine.

VIII. Administration of Curcumin or Curcumin Analogues or Metabolites.

Mostly, it is contemplated liposomes or other colloidal drug-deliveryvehicles containing curcumin or a curcumin analogue would beadministered by intravenous injection, but other routes of parenteraladministration are also conceivable, particularly those that enhancecontact of the liposomes or colloidal drug-delivery vehicles with thetarget tissue. Methods of parenteral administration that can be employedin the present invention also include intraarterial, intramuscular,subcutaneous, intra-tissue, intranasal, intradermal, instillation,intracerebral, intrarectal, intravaginal, intraperitoneal, intratumoral,juxtaposition of tumor and administration directly to the lesion.

In addition, the formulations can also be used as a depot preparation.Such long acting formulations may be administered by implantation at anappropriate site or by parenteral injection, particularly intratumoralinjection or injection at a site adjacent to cancerous tissue.

In alternate embodiments, the formulations of the present invention maybe administered to a mammalian subject, including a human patient, usingbuccal, sublingual, suppository, topical, inhalant or aerosolized routesof administration.

The colloidal drug-delivery system, such as a liposome, containingcurcumin or a curcumin analogue can then be administered to a mammalhaving a tumor or other cancerous growth. Animal studies to date havenever reached an LD₅₀ for free curcumin administration. Oral doses offree curcumin as high as 500 mg/kg and intravenous doses of 40 mg/kghave been administered in rats. (Ireson, 2001). Absorption is minimalafter oral dosing and free curcumin disappears from the circulationusually less than one hour after intravenous dosing. Intravenousadministration of liposomal curcumin has been tolerated by mice at dosesof approximately 40 mg/kg of body weight and no LD50 value has beenreached. In the present invention, when curcumin is encapsulated inliposomes or other colloidal drug-delivery vehicle, any effective amountof the liposomal curcumin may be administered, preferably doses ofapproximately 10-200 mg of curcumin per kg body weight, and mostpreferably 40-100 mg/kg.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples that follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

The surprising utility of liposomal curcumin or curcumin analogues ormetabolites in treating certain cancers is attributable to the potentantiproliferative and proapoptotic effects of curcumin compositions onthose cancer cells. The use of colloidal drug-delivery systems such asliposomes allows the administration of an effective dose of curcumin orcurcumin analogue in vivo for the treatment of cancer. The followingworking examples are illustrative only, and are not intended to limitthe scope of the invention.

The liposomal curcumin of the present invention may be used with one ormore potentiators. As used herein, the term “potentiator” refers to acompound or compounds that accentuate or potentiate a therapeuticresponse, e.g., when the therapeutic relief is cytotoxic activityagainst a cancer cell, the combination of the liposomal curcumin of thepresent invention and the potentiator increases the response above thatof the liposomal curcumin alone. Non-limiting examples of potentiatorsfor use against cancer include, e.g., procodazole, triprolidine,propionic acid, monensin, an anti-sense inhibitor of the RAD51 gene,bromodeoxyuridine, dipyridamole, indomethacin, a monoclonal antibody, ananti-transferrin receptor immunotoxin, metoclopramide,7-thia-8-oxoguanosine,N-solanesyl-N,N′-bis(3,4-dimethoxybenzyl)ethylenediamine, N-[4[(4-fluorphenyl)sulfonyl]phenyl]acetamide, leucovorin, heparin, heparinsulfate, cimetidine, a radiosensitizer, a chemosensitizer, a hypoxiccell cytotoxic agent, muramyl dipeptide, vitamin A, 2′-deoxycoformycin,a bis-diketopiperazine derivative having potentiator activity, dimethylsulfoxide or mixtures thereof. The potentiator may be added inconjunction with the liposomal curcumin. The potentiator may be addedbefore, during or after a dose of liposomal curcumin and may even beconjugated or integrated directly with the liposomal curcumin, eithercovalently or ionically. In one example, the liposomal curcumin and thepotentiator are mixed with a biodegradable resin or matrix that releasesthe liposomal curcumin and the potentiator at the same or differentrates, at the same or disparate times and combinations thereof.

In another non-limiting example, the potentiators for use with theliposomal curcumin to treat a malignant or a non-malignant proliferativedisease, an autoimmune or auto-inflammatory disease or a degenerativedisease. For the treatment of, e.g., Alzheimer's Disease, thepotentiator may be one or more of the following: acetylcholinesteraseinhibitors (e.g., donepezil, galantamine and rivastigmine, which may beprovided in oral form and taken once or twice a day (or less aspotentiators) or even as a transdermal patch); Ginkgo biloba; andN-methyl, D-Aspartate (NMDA) antagonists such as dextromethorphan,dextrorphan, ibogaine, ketamine, nitrous oxide, phencyclidine,memantine, amantadine or tramadol. For potentiation of treatments forParkinson's disease the liposomal curcumin may be provided with, e.g.,dopamine-agonists (e.g., bromocriptine, pergolide, pramipexole,ropinirole, cabergoline, apomorphine, lisuride), monoamine oxidase-B(MAO-B) inhibitors (e.g., rasagiline, selegiline) or L-DOPA (or relatedenzyme inhibitors, e.g., carbidopa, benserazide, tolcapone, entacapone,carbidopa/levodopa and benserazide/levodopa). Non-limiting examples ofpotentiators for the treatment of autoimmune diseases includeimmunosuppressive or anti-inflammatory (e.g., cyclosporine, steroids(e.g., prostaglandins), FK-506, Non-steroidal anti-inflammatory drugs(NSAIDs) (e.g., ibuprofen, diclofenac, aspirin, naproxen), and naturalproducts (e.g., capsaicin, hyssop, ginger, helenalin, willow bark).

In another non-limiting example, the potentiators for use with theliposomal curcumin to treat a parasitic infection include one or moreanti-malarial agents selected from artesiminin, 8-aminoquinoline,amodiaquine, arteether, artemether, artemsinin, artesunate, artesunicacid, artelinic acid, atovoquone, azithromycine, biguanide, chloroquine,chloroquine phosphate, chlorproguanil, cycloguanil, dapsone, desbutylhalofantrine, desipramine, doxycycline, dihydrofolate, reductaseinhibitors, dipyridamole, halofantrine, haloperidol, hydroxychloroquinesulfate, imipramine, mefloquine, penfluridol, phospholipid inhibitors,primaquine, proguanil, pyrimethamine, pyronaridine, quinine, quinidine,quinacrineartemisinin, sulfonamides, sulfones, sulfadoxine, sulfalene,tafenoquine, tetracycline, tetrandine, triazine, salts and derivativesthereof.

In another non-limiting example, the potentiators for use with theliposomal curcumin to treat iron overload or hemochromatosis may includethe common therapy for iron overload or hemochromatosis, periodicphlebotomies while treating with the liposomal curcumin, therebyreducing the number of treatments required.

Example 1

Preparation of Curcumin-Containing Liposomes.

Liposomal curcumin was formulated using the following protocol. Aphospholipid, 1,2-dimyristoyl-sn-glycero-3-phosphocholine(dimyristoylphosphocholine; DMPC) (Avanti Polar Lipids, Alabaster, Ala.35007) was chosen for the liposomal formulation. The phospholipid wassolubilized by dissolving 200 mg of DMPC in 10 ml of t-butanol (FisherScientific) and heating the mixture in a 37° C. water bath for 5minutes. The solution was stored at −20° C. in a container thatprotected the solution from exposure to light.

Curcumin (Sigma) was solubilized by dissolving curcumin in DMSO to afinal concentration of 50 mg/ml. The solution was also aliquoted andstored in a container that protected the solution from exposure tolight.

To combine the phospholipid and curcumin solutions, 10 ml of DMPC int-butanol, 0.4 ml curcumin in DMSO and 90 ml of t-butanol were mixedvery well and aliquoted into small sterile glass vials containing 2.5mls of solution each. The vials of solution were frozen in a dryice-acetone bath and lyophilized overnight using a FTS Systemscorrosion-resistant Freeze-Dryer (Stone Ridge, N.Y.). The dried lipidmixtures were stored at −20° C.

Prior to use, the desired amount of 0.9% NaCl was used to resuspend thelipid mixtures.

Example 2

Curcumin Inhibits Proliferation/Survival of Pancreatic Cells.

Seventy-two hours of exposure to free curcumin inhibited pancreatic cellgrowth of all five lines tested in a concentration-dependent manner.Controls were exposed to 0.1% v/v DMSO. Proliferation and survival ofthe pancreatic cells were assessed by MTT assay, a standard colorimetricassay used to measure cell survival and proliferation (Mosman, 1983).MTT (3-[4,5-cimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) iscleaved by living cells to yield a dark blue formazan product. The coloris quantitated by spectrophotometer and reflects cellular proliferationand survival. As shown in FIGS. 1-5, pancreatic Bxpc-3, Capan-1,Capan-2, HS766-T and ASPC-1 cells were exposed to free curcumin invarying concentrations for a period of 72 hours. The control for eachassay was exposed to 0.1% v/v DMSO.

Example 3

Liposomal Curcumin has Equivalent or Greater Anti-Proliferative Effectsthan Free Curcumin.

The inventors have examined the effect of free curcumin on theproliferation and survival of five pancreatic carcinoma cell lines(Bxpc-3, Capan-1, Capan-2, HS766T, and Aspc-1). The effects of liposomalcurcumin were compared to those of liposomes alone, free curcumin,lyophilized free curcumin and liposomal curcumin. MTTproliferation/survival assay was performed after 72 hours of incubation.

Exposure to curcumin resulted in significant inhibition of proliferationand survival as assessed by MTT assay in all of these cell lines. FIGS.5-10 show graphs of the results of the assays using Bxpc-3, Capan-1,Capan-2, HS766-1 and ASPC-1 cell lines, respectively. In the MTTproliferation/survival assay shown in FIGS. 5-10 the pancreatic celllines were exposed to free curcumin, lyophilized free curcumin(lyophilized curcumin), liposomal curcumin or liposomes without curcumin(liposomes). The MTT assay was performed after pancreatic cells wereexposed for 72 hours to each compound at concentrations ranging from 0to 5 μg/ml. Table I indicates the IC₅₀ and IC₉₀ values calculated fromthe assays of the effects of both free and liposomal curcumin.

TABLE I Inhibitory Concentration of Free vs. Liposomal Curcumin MTTAssay (72 hours incubation) IC₅₀ of IC₉₀ of IC₅₀ of free liposomal IC₉₀of free liposomal Name of Cells curcumin curcumin curcumin curcuminBXPC-3 2 μg/ml 2 μg/ml 5 μg/ml 5 μg/ml 5.4 μM 5.4 μM 13.5 μM 13.5 μMCAPAN-1 2 μg/ml 0.75 μg/ml 5 μg/ml 2.5 μg/ml 5.4 μM 2 μM 13.5 μM 6.75 μMCAPAN-2 17 μg/ml 14 μg/ml 35 μg/ml 35 μg/ml 46 μM 37.8 μM 94.5 μM 94.5μM HS766-T 2.6 μg/ml 2.5 μg/ml 8 μg/ml 9 μg/ml 7 μM 6.75 μM 21.6 μM 24μM ASPC-1 4 μg/ml 4 μg/ml 10 μg/ml 10 μg/ml 10.8 μM 10.8 μM 27 μM 27 μM

The 50% inhibitory concentration (IC₅₀) of free curcumin varied fromapproximately 5 μm in Capan-1 and BxPC-3 cells to about 46 μM in Capan-2cells. The 50% inhibitory concentration (IC₅₀) of liposomal curcuminvaried from approximately 2 μm in Capan-1 cells to about 38 μM inCapan-2 cells. The 90% inhibitory concentration (IC₉₀) of free curcuminvaried from approximately 14 μm in Capan-1 and BxPC-3 cells to about 95μM in Capan-2 cells. The 90% inhibitory concentration (IC₉₀) ofliposomal curcumin varied from approximately 7 μm in Capan-1 cells toabout 95 μM in Capan-2 cells. The results demonstrate that the growthinhibitory effects of liposomal curcumin were approximately equal to orbetter than that of free curcumin. Empty liposomes had minimalgrowth/survival suppressive effects.

Example 4

The Growth Inhibitory Effects of Liposomal Curcumin are Irreversible.

Pancreatic cell recovery of proliferation and survival after exposure toliposomal curcumin was determined for Bxpc-3, Capan-1, Capan-2, HS766-1and ASPC-1 cell lines, respectively. Pancreatic cells were grown onstandard media. One control was untreated pancreatic cells. Anothercontrol was pancreatic cells treated with 0.1% DMSO, the solvent used todissolve free curcumin. In addition to the controls, pancreatic cellswere treated with empty liposomes or liposomal curcumin for 72 hours.The concentrations of liposomal curcumin used were approximately theIC₅₀ and IC₉₀ for each cell line (as determined in Example 3). Afterexposure, cells were replated in fresh media and allowed to recover inthe absence of curcumin or liposomes. Recovery of proliferation/survivalwas assessed after an additional 72 hours. The cells were assayed by MTTassay 72 hours after replating and the results are shown in FIGS. 11-15.

The sets of bars of the graph in FIG. 11 (left to right) correspond tocontrol BxPC-3 cells followed by BxPC-3 cells exposed to empty liposomesor to 5 μg/ml (13.5 μM) liposomal curcumin, the latter being the IC₉₀concentration of liposomal curcumin for BxPC-3 cells. The left bar ofeach pair corresponds to assay results after 72 hours of treatment. Theright bar corresponds to assay results after 72 hours of treatment plus72 hours recovery on fresh media without treatment.

The sets of bars of the graph in FIG. 12 (left to right) correspond tocontrol Capan-1 cells followed by Capan-1 cells exposed to emptyliposomes or to 2.5 μg/ml (6.75 μM liposomal curcumin), the latter beingthe IC₉₀ concentration of liposomal curcumin for Capan-1 cells. The leftbar of each pair corresponds to assay results after 72 hours oftreatment. The right bar corresponds to assay results after 72 hours oftreatment plus 72 hours recovery on fresh media without treatment.

In FIG. 13, the sets of bars of the graph (left to right) correspond tocontrol Capan-2 cells followed by Capan-2 cells exposed to emptyliposomes, 14 μg/ml (37.8 μM) liposomal curcumin, empty liposomes, and35 μg/ml (94.5 μM) liposomal curcumin. 37.8 μM represents the IC₅₀ and94.5 μM represents the IC₉₀ for liposomal curcumin for Capan-2 cells.(The amount of empty liposomes is equivalent to the amount of liposomalmaterial in the corresponding experiments with liposomal curcumin, i.e.the IC₅₀ and IC₉₀ levels, respectively.) The left bar of each paircorresponds to assay results after 72 hours of treatment. The right barcorresponds to assay results after 72 hours of treatment plus 72 hoursrecovery on fresh media without treatment.

In FIG. 14, the sets of bars of the graph (left to right) correspond tocontrol HS766-T cells followed by HS766-T cells exposed to emptyliposomes, 2.5 μg/ml (6.75 μM) liposomal curcumin, empty liposomes, and9 μg/ml (24 μM) liposomal curcumin. 6.75 μM represents the IC₅₀ and 24μM represents the IC₉₀ for liposomal curcumin for HS766-T cells. (Theamount of empty liposomes is equivalent to the amount of liposomalmaterial in the corresponding experiments with liposomal curcumin, i.e.the IC₅₀ and IC₉₀ levels, respectively.) The left bar of each paircorresponds to assay results after 72 hours of treatment. The right barcorresponds to assay results after 72 hours of treatment plus 72 hoursrecovery on fresh media without treatment.

In FIG. 15, the sets of bars of the graph (left to right) correspond tocontrol ASPC-1 cells followed by ASPC-1 cells exposed to emptyliposomes, 4 μg/ml (10.8 μM) liposomal curcumin, empty liposomes, and 10μg/ml (27 μM) liposomal curcumin. 4 μM represents the IC₅₀ and 27 μMrepresents the IC₉₀ for liposomal curcumin for ASPC-1 cells. (The amountof empty liposomes is equivalent to the amount of liposomal material inthe corresponding experiments with liposomal curcumin, i.e. the IC₅₀ andIC₉₀ levels, respectively.) The left bar of each pair corresponds toassay results after 72 hours of treatment. The right bar corresponds toassay results after 72 hours of treatment plus 72 hours recovery onfresh media without treatment.

As shown by FIGS. 11-15, there was a concentration-dependent loss ofability to recover, indicating that the effect of liposomal curcumin wasnot merely cytostatic, but rather resulted in induction of apoptosis orcell death.

Example 5

Both Free and Liposomal Curcumin Induce Apoptosis in Pancreatic Cancer.

Apoptosis of pancreatic cells was assessed by Annexin-V/Proopidiumiodide staining (FACS analysis) after 72 hours of exposure to a DMSOcontrol, empty liposomes, free curcumin or liposomal curcumin. Apoptosisassay was performed using either pancreatic BxPC-3 or HS766-T cells. Thecells were exposed to either a control of 0.1% DMSO, empty liposomes,free curcumin or liposomal curcumin for 72 hours. Free curcumin andliposomal curcumin were administered in concentrations equal to eitherthe IC₅₀ or IC₉₀ of liposomal curcumin. After incubation for 72 hours,the cells were assayed using Annexin-V/Proopidium iodide staining (FACSanalysis) to determine how much apoptosis had occurred.

For FIG. 16, bars for free and liposomal curcumin on the left side ofthe graph correspond to a concentration of curcumin if 5 μM, which isthe IC₅₀ for liposomal curcumin in BxPC-3 cells. Bars for fee andliposomal curcumin on the right side of the graph correspond to aconcentration of curcumin of 13.5 μM, which is the IC₉₀ for liposomalcurcumin in BxPC-3 cells.

For FIG. 17, bars for free and liposomal curcumin on the left side ofthe graph correspond to a concentration of curcumin if 7 μM, which isthe IC₅₀ for liposomal curcumin in HS766-T cells. Bars for fee andliposomal curcumin on the right side of the graph correspond to aconcentration of curcumin of 25 μM, which is the IC₉₀ for liposomalcurcumin in HS766-T cells.

As can be seen in FIGS. 16 and 17, there was dose-related apoptosisafter treatment with either free curcumin or liposomal curcumin.Apoptotic induction by liposomal curcumin greater than that of freecurcumin at both the IC₅₀ and IC₉₀ for liposomal curcumin. Emptyliposomes had no significant apoptotic effect. At the IC₉₀ for liposomalcurcumin (as determined in Example 3) liposomal curcumin induced 73% to93% apoptosis after 72 hours.

Example 6

Liposomal Curcumin has Anti-Proliferative Effects on Melanoma Cells.

Liposomal curcumin was shown to have antiproliferative and apoptoticeffects on two melanoma cell lines, HT-144 and A375, approximatelyequivalent to that of free curcumin. Cells were grown in the presence0.1% DMSO (control), free curcumin, empty liposomes or liposomalcurcumin for 72 or 96 hours. Free curcumin and liposomal curcumin wereadministered at concentrations of 10 μM, 20 μM and 40 μM. The cells werethen assessed for cell proliferation and survival using an MTT assay.FIG. 18 shows the results of the assays for A375 cells cultured in thepresence of free curcumin, empty liposomes or liposomal curcumin for 72hours, as a percentage of growth versus the control cells. FIG. 19 showsthe results of the assays for HT-144 cells cultured in the presence offree curcumin, empty liposomes or liposomal curcumin for 96 hours, as apercentage of growth versus the control cells.

Example 7

Liposomal Curcumin has Anti-Proliferative Effects on Breast CancerCells.

Liposomal curcumin was shown to inhibit the proliferation of cells ofthe breast cancer cell line MCF-7. The effects of liposomal curcuminwere compared to those of liposomes alone and to free curcumin. Twoforms of MCF-7 breast cancer line were used—one resistant to Adriamycinand one which is sensitive to Adriamycin (wild type (wt)). MTTproliferation/survival assay was performed after 48 hours of incubation.The results, shown in FIGS. 20 and 21, demonstrate that liposomalcurcumin inhibits both the Adriamycin-resistant and the sensitive line.Its effects are equivalent to those of free curcumin.

Example 8

The present invention can be used for the treatment of cancers. Inparticular, the present invention may be used for the treatment ofpancreatic cancer.

To treat a human patient or other mammalian subject having pancreaticcancer an effective amount of curcumin or curcumin analogue encapsulatedin a colloidal drug delivery system is administered parenterally to theanimal or patient. Lower limits on the amount of therapeutic agent to beadministered is the amount required to elicit a therapeutic effect asdetermined based upon animal pharmacology and early phase clinicaltrials in humans, both of which are standard activities and practices inthe pharmaceutical industry. The upper limits on the amount oftherapeutic agent to be administered is determined based on the toxicityof the therapeutic agent used. One of skill in the art could identifyand quantify variables that would define the toxicity associated withthe colloidal drug delivery system containing curcumin or curcuminanalogue encapsulated curcumin or curcumin analogue.

Example 9

To treat a human patient or other mammalian subject having breast canceran effective amount of curcumin or curcumin analogue encapsulated in acolloidal drug delivery system is administered parenterally to theanimal or patient. Lower limits on the amount of therapeutic agent to beadministered is the amount required to elicit a therapeutic effect asdetermined based upon animal pharmacology and early phase clinicaltrials in humans, both of which are standard activities and practices inthe pharmaceutical industry. The upper limits on the amount oftherapeutic agent to be administered is determined based on the toxicityof the therapeutic agent used. One of skill in the art could identifyand quantify variables that would define the toxicity associated withthe colloidal drug delivery system containing curcumin or curcuminanalogue encapsulated curcumin or curcumin analogue.

Example 10

To treat a human patient or other mammalian subject having melanoma aneffective amount of curcumin or curcumin analogue encapsulated in acolloidal drug delivery system is administered parenterally to theanimal or patient. Lower limits on the amount of therapeutic agent to beadministered is the amount required to elicit a therapeutic effect asdetermined based upon animal pharmacology and early phase clinicaltrials in humans, both of which are standard activities and practices inthe pharmaceutical industry. The upper limits on the amount oftherapeutic agent to be administered is determined based on the toxicityof the therapeutic agent used. One of skill in the art could identifyand quantify variables that would define the toxicity associated withthe colloidal drug delivery system containing curcumin or curcuminanalogue encapsulated curcumin or curcumin analogue.

Example 11

The present invention can be used for the prevention of cancers. Inparticular embodiments, the present invention may be used for theprevention of pancreatic cancer, breast cancer or melanoma.

To serve as a cancer preventative or prophylactic, an effective amountof curcumin or curcumin analogue encapsulated in a colloidal drugdelivery system is administered parenterally to the animal or humanpatient at risk for developing a specific cancer. Lower limits on theamount of therapeutic agent to be administered is the amount required toelicit a therapeutic effect as determined based upon animal pharmacologyand early phase clinical trials in humans, both of which are standardactivities and practices in the pharmaceutical industry. The upperlimits on the amount of therapeutic agent to be administered isdetermined based on the toxicity of the therapeutic agent used. One ofskill in the art could identify and quantify variables that would definethe toxicity associated with the colloidal drug delivery systemcontaining curcumin or curcumin analogue encapsulated curcumin orcurcumin analogue.

Example 12

Conventional and sterically-stabilized (PEGylated) liposomes werestudied for their efficiency of curcumin incorporation. The results areindicated in Table 2. The numbers in the composition and charge columnrepresent the weight to weight (w/w) ratio of the respective lipids inthe formulation in liposomes containing more than one type of lipid.

The curcumin encapsulation efficiency was determined after the liposomeswere reconstituted with saline using a pipette to produce 10 μl ofsuspension. The suspension was checked by microscopy at 400× in bothvisible light and fluorescence light. “No” indicates that crystals andneedles of free curcumin remained. “Yes” indicates no crystals andneedles of free curcumin remained. Liposomes that could be reconstitutedwith saline at room temperature or by bath sonication at 37° C. for 5 to10 minutes were classified “Yes” for ease of handling.

The tendency of the liposomal particles to self-aggregate was checkedunder a microscope after reconstitution in saline. “No” indicates thatself-aggregation was not observed; “Yes” indicates that it was.

The data found in the growth inhibition column of Table 2 was generatedusing an MTT assay to assess growth and survival of tested cells. Thepancreatic cancer cells Bxpc-3 were plated in 96-well plates overnightand 7.5 μg/ml of either free or liposomal curcumin was added andincubated for 72 hours. MTT dye (5 mg/ml in PBS) was added and incubatedfor 4 hours. At the end of the incubation, the amount of formazan formedwas read at 570 nm and the results are as indicated.

Liposomal toxicity was also assessed. The pancreatic cancer cells Bxpc-3were plated in 96-well plates overnight. Empty liposomes not containingcurcumin were supplied in a quantity equivalent to that of thecurcumin-containing liposomes used in the growth inhibition study. Theempty liposomes were added to the Bxpc-3 cells and incubated for 72hours. MTT dye (5 mg/ml in PBS) was added and incubated for 4 hours andat the end of incubation, the amount of the formazan formed was read at570 nm. The results are as indicated in the column labeled “liposometoxicity.”

In Table II, “PC” denotes L-α-phosphatidylcholine (egg, chicken) and“PS” denotes L-α-phosphatidylserine (brain, porcine)(sodium salt).

TABLE II Summary of characteristics of various liposomal-curcuminformulations. Curcumin Composition and charge encapsulates Ease ofLiposome self Growth Liposome w/w efficiency Handling AggregationInhibition Toxicity Conventional Liposome Neutral PC Low PC:chol 9:1 LowPC:chol 7:3 Low DMPC High Yes Yes  79 ± 1.6 13 DMPC:Chol 9:1 High YesYes  89 ± 0.1 17 DOPC Low DPPC High No DPPC:Chol (90:11.5) High No YesPositively charged DMPC:SA 9:1 Low Negatively charged DMPC:DMPG 7:3 LowDMPC:DMPG 9:1 High Yes No 81 ± 4 11 DMPC:Chol:DMPG 8:1:1 High Yes Yes 65 ± 0.4 17 DPPC:DMPG 7:3 Low DPPC:DMPG 9:1 High No No 81 ± 4 14DPPC:Chol:DMPG 8:1:1 High No Yes   67 ± 0.06 14 DPPC:DPPG 7:3 LowDOPC:DMPG 7:3 Low DOPC:DMPG 9:1 Low PC:PS:Chol 7:3:1 LowSterically-Stabilized Liposome Pegylated and Negatively chargeDMPC:DMPE-PEG-2000 90:10 Low DMPC:DMPE-PEG-2000 95:5 High Yes No 58.5 ±4.5 22.5 DMPC:DMPE-PEG-2000 97:3 Low DMPC:Chol:DMPE-PEG-2000 High Yes No85 ± 1 13.5 90:10:05 DMPC:DSPE-PEG-2000 95:5 High No No 64 ± 4 20DMPC:Chol:DSPE-PEG-2000 High No No 69 ± 9 19 90:10:05

As shown by the results of Table 2, the optimal formulations ofliposomal curcumin included DMPC/DMPG (9:1) and DMPC/CHOL/DMPE-PEG-2000(90:10:5). The latter is a pegylated formulation. These formulationswere chosen as being optimal among the compositions studied on the basisof their properties including (i). encapsulation efficiency, (ii) easeof handling, (iii) liposome self-aggregation, (iv) growth inhibition and(v) level of toxicity of empty liposomes.

Example 13

The optimal curcumin-to-lipid ratio (weight-to weight) was determinedfor several liposomal curcumin formulations made using DMPC. The ease ofhandling was determined by determining whether or not the lipid-curcuminmixture could be readily dissolved in t-butanol without DMSO. AlthoughDMSO had been used in previous studies, t-butanol may be preferred forcertain pharmaceutical uses. The degree to which free curcuminassociated with the lipid without the formation of needles or crystalsof free curcumin was assessed by microscopy. The optimalcurcumin-to-DMPC ratio among the ratios tested was 1:10.

TABLE III Optimization of the Curcumin-to-Lipid ratio in LiposomalCurcumin Formulations Curcumin:DMPC Ease of (w/w) HandlingCurcumin:Lipid Association 1:10 Yes Curcumin completely associated withliposome 1:7.5 Yes Very few curcumin crystals 1:5 No Few curcumincrystals

Example 14

A variety of lipids and lipid mixtures were assayed for their ability toform liposomes encapsulating curcumin and the results are disclosed inTable IV. The t-butanol was pre-warmed for 10 min at 37° C. Lipids wereweighed out in the appropriate quantities and placed in glass vials and25 ml of pre-warmed t-butanol is added to each vial. The mixtures werevortexed and sonicated in a bath sonicator (Branson 2200, Danbury,Conn.) for 5 minutes. The curcumin was weighed out and 5 mg of curcuminwas added to each vial. The mixtures were vortexed in the sonicator bathfor 5 minutes until the curcumin was completely dissolved. The vialswere frozen in a dry ice-acetone bath and lyophilized for 24 hours in aFreeze Dry System (Freezone 4.5, Labconco). The vials were stored at−20° C. in a container that protected the composition from exposure tolight. The lyophilized powder was subsequently warmed to roomtemperature for 10 minutes and reconstituted with saline (0.9% NaCl) at4 mg/ml using a bath sonicator for 5 minutes. From each vial, 10 μl ofeach formulation was placed on a glass slide, covered with a cover slideand checked for appearance under a fluorescence microscope.

The numbers after the abbreviations in the Lipid Composition column ofTables IV and V indicate the proportions of lipids used in the lipidmixtures; ratios in brackets are lipid to curcumin ratios (w/w). TableIV notes the appearance of the lyophilizate for each formulation, theease of the reconstitution in saline and the appearance of thereconstituted composition under a microscope. The reconstitutedliposomes were centrifuged at 1000 rpm for 10 minutes, and theappearance of the pellet and supernatant was noted. Free curcumin andlarger liposomes will tend to accumulate in the pellet. Experimentalobservations are listed in the final column of Table IV. “SA” denotesstearylamine. In Table IV, “PC” denotes L-α-phosphatidylcholine (egg,chicken) and “PS” denotes L-α-phosphatidylserine (brain, porcine)(sodium salt). As used herein, any characterization that a formulationwas “poor” or that it performed “poorly” in a particular application isnot an indication that the formulation is unsuitable for use, but merelyreflects that the formulation was less preferred than other formulationsunder the assay conditions tested.

TABLE IV Encapsulation Efficiency of Various Conventional LiposomalCurcumin Formulations Part I Lipid Appearance after Ease of Appearanceafter Composition Lyophilization Reconstitution Centrifugation CommentsDMPC/DMPG Fluffy powder Easy to reconstitute; Small pellets; Curcumin is7:3 very few crystals and unclear encapsulated needles; adequatesupernatant well liposomes PC/PS/Chol Unevenly Easy to reconstitute;Small pellets; Curcumin is 7:3:1 distributed film crystals and needlesunclear encapsulated are present supernatant poorly DMPC/SA Red cake Onaddition of SA to Dark orange Unacceptable; a 9:1 the solution it turnedcolor pellets chemical reaction red and formed a between SA andsolid-colored cake curcumin appears to have occurred DPPC Fluffy powderHard to reconstitute; Quite clear Difficult to supernatant handleUniform pellets DPPC/DPPG Fluffy powder Hard to reconstitute; Smallpellets; Difficult to few crystals and unclear handle needlessupernatant DPPC/DMPG Fluffy powder few crystal and Small pellets;Curcumin is 7:3 needles unclear encapsulated supernatant well DOPC/DMPGUnevenly Easy to reconstitute; Small pellets; Curcumin is 7:3distributed film few crystal and unclear encapsulated needlessupernatant poorly DMPC [10:1] Fluffy powder Nice liposomes Quite clearOptimal supernatant; curcumin uniform pellets encapsulation DMPC [7.5:1]Fluffy powder Nice liposmoes and Quite clear Curcumin is very fewcrystals supernatant; encapsulated uniform pellets poorly DMPC [5:1]Fluffy powder Nice liposomes and Quite clear Curcumin is few crystalssupernatant; encapsulated uniform pellets poorly DOPC [10:1] Evenly Easyto reconstitute; Quite clear Optimal distributed film few crystals andsupernatant; curcumin needles uniform pellets encapsulation DOPC [7.5:1]Evenly Easy to reconstitute; Quite clear Curcumin is distributed filmcrystals and needles supernatant; encapsulated uniform pellets poorlyDOPC [5:1] Evenly Easy to reconstitute; Quite clear Curcumin isdistributed film many crystals and supernatant; encapsulated needlesuniform pellets poorly PC Unevenly Uneven-sized, self- Quite clearCurcumin is distributed film aggregated liposomes; supernatant;encapsulated crystals uniform pellets poorly PC/Cholesterol UnevenlyUneven-sized, self- Quite clear Curcumin is 9:1 distributed filmaggregated liposomes; supernatant; encapsulated crystals uniform pelletspoorly PC/Cholesterol Unevenly Uneven-sized, self- Quite clear Curcuminis 7:3 distributed film aggregated liposomes; supernatant; encapsulatedcrystals uniform pellets poorly

Table V lists the observed encapsulation properties of several otherlipid compositions. The comments indicate the optimal and preferredliposomal compositions among those tested. The protocol used to obtainthe data in Table V is a follows:

Material:

-   -   DMPC; DPPC, DMPG, DOPC, PC, Cholesterol (Avanti Polar Lipids,        Alabaster, Ala. 35007)    -   T-butanol (Sigma)    -   Dry ice    -   Acetone (Sigma)    -   Heat-resistant glass vial    -   Curcumin (Sigma)        Equipment:    -   1. Freeze Dry System (Labconco, Fisher Scientific)    -   2. Sonicator (Branson 2200, Danbury, Conn.)        Method:    -   1. Pre-warm T-butanol for 10 minutes at 37° C.    -   2. Weigh out DPPC: 45 mg and DMPG 5 mg (9:1); DMPC 50 mg; DOPC:        50 mg; PC 45 mg and Cholesterol 5 mg (9:1); DMPC 45 mg and DMPG        5 mg (9:1); DOPC 45 mg and DMPG 5 mg (9:1), and put into 6 glass        vials.    -   3. Add 25 ml pre-warmed T-butanol to each vial. Vortex and        sonicate at a bath sonicator for 5 minutes.    -   4. Weigh out curcumin 6×5 mg (5 mg/vial) add to each vial above        vortex and bath sonicate for 5 min until curcumin completely        dissolved.    -   5. Aliquot and freeze vials at a Dry ice-acetone bath and        lyophilize for 24 hours at a Freeze Dry System (Freezone 4.5,        Labconco). Store at −20° C.    -   6. Warm up the liposomal curcumin powder at room temperature for        10 minutes. Reconstitute with saline (0.9% NaCl) at 4 mg/ml,        bath sonicate for 5 minutes.    -   7. Take 10 μl of each formulation vial and put on a glass slide        and covered with a cover slide and check under a fluorescence        microscope.    -   8. Powder is stored in a container that protects it from        exposure to light.

The results are as follows:

TABLE V Encapsulation Efficiency of Various Conventional LiposomalCurcumin Formulations Part II Lipid Appearance after Ease of Appearanceafter Composition Lyophilization Reconstitution Centrifugation CommentDPPC/DMPG Nice fluffy Hard to Large volume of GOOD 9:1 powderreconstitute with yellow precipitations saline; very nice (L-cur), Verysmall liposomes volume of orange- colored precipitate (Free curcumin).DMPC Nice fluffy Easy to Large volume of GOOD powder reconstitute withyellow precipitations saline at 37° C.; (L-cur), Very small very nicevolume of orange- liposomes colored precipitate (Free curcumin). DOPCUnevenly Easy to Small volume of POOR distributed film reconstitute withyellow precipitations saline at room (L-cur), Large volume temperature;of orange-colored needles and precipitate (Free crystals presentcurcumin). PC/Cholesterol Unevenly Easy to Small volume of POOR 9:1distributed film reconstitute with yellow precipitations saline; needles(L-cur), Large volume and crystals of orange-colored present precipitate(Free curcumin). DMPC/DMPG Nice fluffy Easy to Very large volume ofOPTIMAL 9:1 powder reconstitute with yellow precipitations saline at 37°C.; (L-cur), trace of very nice orange-colored liposomes precipitate(Free curcumin). DOPC/DMPG Unevenly Easy to Small volume of POOR 9:1distributed film reconstitute with yellow precipitations saline at room(L-cur), Large volume temperature; of orange-colored needles andprecipitate (Free crystals present curcumin).

Example 15

Several preferred formulations of pegylated liposomes where prepared andtested according to the following protocol:

Material:

-   -   DMPC; DMPE peg 2000; (Avanti Polar Lipids, Alabaster, Ala.        35007)    -   T-butanol (Sigma)    -   Dry ice    -   Acetone (Sigma)    -   Heat-resistant glass vial    -   6: Curcumin (Sigma)        Equipment:    -   3. Freeze Dry System (Labconco, Fisher)    -   4. Sonicator (Branson 2200, Danbury, Conn.)        Method:    -   1. Pre-warm T-butanol for 10 minutes at 37° C.    -   2. Weigh out DMPC 45 mg and DMPE peg 2000 5 mg (90:10); DMPC        47.5 mg and DMPE peg 2000 2.5 mg (95:5); DMPC 48.5 mg and DMPE        peg 2000 1.5 mg (97:3), and put into 3 glass vials.    -   3. Add 25 ml pre-warmed t-butanol to each vial. Vortex and        sonicate in a bath sonicator for 5 minutes.    -   4. Weigh out curcumin 3×5 mg (5 mg/vial) ads to each vial above        vortex and bath sonicate for 5 minutes until curcumin completely        dissolved.    -   5. Aliquot and freeze vials at a Dry ice-acetone bath and        lyophilize for 24 hours at a Freeze Dry System (Freezone 4.5,        Labconco). Store at −20° C.    -   6. Warm up the Liposomal Curcumin powder at room temperature for        10 minutes. Reconstitute with saline (0.9% NaCl) at 4 mg/ml,        bath sonicate for 5 minutes.    -   7. Take 10 μl of each formulation vial and put on a glass slide        and covered with a cover slide and check under a fluorescence        microscope.    -   8. Powder is stored in a container that protects it from        exposure to light.

The formulations were reconstituted and checked by microscopy, as above.The formulations were assessed for ease of handling, reconstitution andencapsulation characteristics as in the examples above. The results areshown in Table VI and VII. Among the formulations tested in Table VI,DMPC:DMPE-PEG-2000 95:5 (w/w) was found to have optimal properties. Asused herein, any characterization that a formulation was “poor” or thatit performed “poorly” in a particular application is not an indicationthat the formulation is unsuitable for use, but merely reflects that theformulation was less preferred than other formulations under the assayconditions tested.

TABLE VI Optimization of Lipid/PEGylated Lipid Ratios in LiposomalCurcumin Formulations Curcumin DMPC:DMPE Ease of encapsulation PEG-2000(w/w) Handling Ease of Reconstitution efficiency Comments 90:10 YesLiposomes present; uneven ++ POOR size particles; crystals and needlespresent 95:5 Yes Nice liposomes; uneven ++++ OPTIMAL size particles;very few crystals 97:3 Yes Liposomes present; uneven ++++ GOOD sizeparticles; a few crystals and a few self-aggregated liposomes

Of the formulations tested in Table VII, the optimal formulation wasDMPC/Cholesterol/DMPE-PEG-2000, 90:10:5 (w/w). The liposomes did notappear to self-aggregate and tended to remained in suspension duringcentrifugation. Very little free curcumin was observed in thisformulation.

TABLE VII Encapsulation Efficiency of Various Sterically-Stabilized(PEGylated) Liposomal Curcumin Formulations Lipid Ease of Ease ofAppearance after composition Handling Reconstitution centrifugationComments DMPC/Chol Yes Nice liposome; Nice pellets; clear POOR, 9:1(w/w) even size particles; supernatant; some liposomes self-self-aggregated free cucumin aggregate liposomes observed DMPC/Chol/ YesNice liposomes, Liposomes remain OPTIMAL DMPE-PEG-2000 easy to handle;suspended; 90:10:5 (w/w) uneven size small pellets; particles verylittle free curcumin observed DMPC/Chol/ No Nice liposomes, Liposomesremain GOOD DSPE-PEG-2000 easy to handle; suspended; 90:10:5 (w/w)uneven size small pellets; particles some free curcumin observedDMPC/DMPE- Yes Nice liposomes, Liposomes remain GOOD, PEG-2000 easy tohandle; suspended; but empty 95:5 (w/w) uneven size small pellets;liposomes are particles very little free toxic to curcumin observedMiapaca-2 cells DMPC/DSPE- No Nice liposomes, Liposomes remain GOOD,PEG-2000 easy to handle; suspended; but empty 95:5 (w/w) uneven sizesmall pellets; liposomes are particles very little free toxic tocurcumin observed Miapaca-2 cells

Example 16

Various conventional liposomal curcumin formulations were tested fortheir effects on cancer cell proliferation and survival using the MTTassay described in previous examples. FIGS. 22-24 compare the effects ofliposomal curcumin and empty liposomes on the growth of several strainsof human pancreatic cancer cells. FIG. 22 assesses BXPC-3 cells, FIG. 23assesses MiaPaCa-2 cells, and FIG. 24 assesses ASPC-1 cells.

In FIGS. 22-24, DPPC/DMPG and DPPC/DMPG/Cur have lipid ratios of 9:1 andthe liposomal curcumin formulation has a lipid to curcumin ratio of10:1. The liposomes were prepared by the following protocol:

All curcumin-containing formulations have a lipid to curcumin ratio of10:1. The DPPC/DMPG and DMPC/DMPG formulations have lipid ratios of 9:1.The liposomes were prepared by the following protocol:

Material:

-   -   DPPC; DMPG; DMPC; (Avanti Polar Lipids, Alabaster, Ala. 35007)    -   T-butanol (Sigma)    -   Dry ice    -   Acetone (Sigma)    -   Heat-resistant glass vial    -   Curcumin (Sigma)        Equipment:    -   1. Freeze Dry System (Labconco, Fisher)    -   2. Sonicator (Branson 2200, Danbury, Conn.)        Method:        Pre-warm T-butanol for 10 minutes at 37° C.    -   1. Weigh out appropriate quantities of lipid to achieve the        indicated lipid ratios for a total of 50 mg of lipid, and put        into 5 glass vials.    -   2. Add 25 ml pre-warmed T-butanol to each vial. Vortex and        sonicate in a bath sonicator for 5 minutes.    -   3. Weigh out curcumin 5×5 mg (5 mg/vial) add to each vial above        vortex and bath sonicate for 5 min until curcumin completely        dissolved.    -   4. Aliquot and freeze vials at a Dry ice-acetone bath and        lyophilize for 24 hours at a Freeze Dry System (Freezone 4.5,        Labconco). Store at −20° C.    -   5. Warm up the Liposomal Curcumin powder at room temperature for        10 minutes. Reconstitute with saline (0.9% NaCl) at 4 mg/ml,        bath sonicate for 5 minutes.    -   6. Take 10 μl of each formulation vial and put on a glass slide        and covered with a cover slide and check under a fluorescence        microscope.    -   7. Powder is stored in a container that protects it from        exposure to light.

For each figure, the pancreatic cancer cells were grown in 96-wellplates overnight. Free curcumin and liposomal curcumin was added to themedia at concentrations ranging from 1 to 10 μg/ml. Empty liposomes wereadded at concentrations equivalent to the lipid concentration found inthe liposomal curcumin formulations. The cells were incubated for 72hours after the addition of free curcumin or liposomes and then cellviability was assessed using the MTT assay. The results shown by thebars are the mean values for three different experiments. The error barsindicate the standard deviation for each set of experiments.

Example 17

Various PEGylated liposomal curcumin formulations were tested for theireffects on cancer cell proliferation and survival using the MTT assaydescribed in previous examples. FIGS. 25 and 26 compare the effects ofliposomal curcumin and empty liposomes on the growth of two strains ofhuman pancreatic cancer cells. FIG. 25 assesses BXPC-3 cells and FIG. 26assesses MiaPaCa-2 cells.

In FIGS. 25 and 26, liposomes containing curcumin contain a lipid tocurcumin ratio of 10:1. The liposomes were prepared by the same protocolas in Example 16 except that the lipids used included DMPE-PEG-2000;DSPE-PEG-2000 and Cholesterol from Avanti Polar Lipids, Alabaster, Ala.35007.

For each figure, the pancreatic cancer cells were grown in 96-wellplates overnight. Free curcumin and liposomal curcumin was added to themedia at concentrations ranging from 1 to 10 μg/ml. Empty liposomes wereadded at concentrations equivalent to the lipid concentration found inthe liposomal curcumin formulations. The cells were incubated for 72hours after the addition of free curcumin or liposomes and then cellviability was assessed using the MTT assay. The results shown by thebars are the mean values for three different experiments. The error barsindicate the standard deviation for each set of experiments.

Example 18

In another example, a composition for the efficient loading of curcumin,includes an amount of a curcuminoid:liposome complex effective to loadcurcumin into the liposome, wherein the curcuminoids comprise between 2to 9 weight percent of the total composition and the curcuminoids arenatural or synthetic. For example, the liposome may be PEGylated. In oneembodiment, the composition is a DMPC/Chol/DMPE-PEG-2000 liposome at aratio of between 90:10:2 (w/w) to 90:10:9 (w/w) and the curcuminoids; aDMPC/Chol/DSPE-PEG-2000 liposome at a ratio of between 90:10:2 (w/w) to90:10:9 (w/w) and the curcuminoids; a DMPC/DMPE-PEG-2000 liposome at aratio of between 90:10:2 (w/w) to 90:10:9 (w/w) and the curcuminoids; ora DMPC/DSPE-PEG-2000 liposome at a ratio of between 90:10:2 (w/w) to90:10:9 (w/w) and the curcuminoid. The curcuminoid may be administeredin a dose of from about 0.01 mg/kg of the individual's body weight toabout 500 mg/kg of the individual's body weight. The curcumin may beselected from the group consisting of Ar-tumerone, methylcurcumin,demethoxy curcumin, bisdemethoxycurcumin, sodium curcuminate,dibenzoylmethane, acetylcurcumin, feruloyl methane, tetrahydrocurcumin,1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione (curcumin1),1,7-bis(piperonyl)-1,6-heptadiene-3,5-dione (piperonyl curcumin)1,7-bis(2-hydroxy naphthyl)-1,6-heptadiene-2,5-dione (2-hydroxylnaphthyl curcumin) and 1,1-bis(phenyl)-1,3,8,10undecatetraene-5,7-dione.

Example 19

The present invention includes a method of treating a malignant or anon-malignant proliferative disease, an autoimmune or auto-inflammatorydisease or a degenerative disease comprising providing a patient in needthereof with an effective amount of a curcuminoid:PEGylated-liposomeeffective to load curcumin into the liposome, wherein the liposomecomprises a ratio of liposome to PEG has between 2 to 9 weight percentof the total composition and the curcuminoids are natural or syntheticand/or the use of a medicament with the above characteristics for thetreatment of malignant or a non-malignant proliferative disease, anautoimmune or auto-inflammatory disease or a degenerative disease. Inone specific embodiment, the composition used in the method is aDMPC/Chol/DMPE-PEG-2000 liposome at a ratio of between 90:10:2 (w/w) to90:10:9 (w/w) and the curcuminoids; a DMPC/Chol/DSPE-PEG-2000 liposomeat a ratio of between 90:10:2 (w/w) to 90:10:9 (w/w) and thecurcuminoids; a DMPC/DMPE-PEG-2000 liposome at a ratio of between90:10:2 (w/w) to 90:10:9 (w/w) and the curcuminoids; or aDMPC/DSPE-PEG-2000 liposome at a ratio of between 90:10:2 (w/w) to90:10:9 (w/w) and the curcuminoids. For example, the curcuminoid isadministered in a dose of from about 0.01 mg/kg of the individual's bodyweight to about 500 mg/kg of the individual's body weight.

TABLE VIII Lipid composition Potentiator Anti-Cancer DMPC/Chol/0.1-1,000 mg procodazole, triprolidine, propionic acid, monensin, anDMPE-PEG-2000 anti-sense inhibitor of the RAD51 gene, 90:10:5 (w/w);bromodeoxyuridine, dipyridamole, indomethacin, a DMPC/Chol/ monoclonalantibody, an anti-transferrin receptor DSPE-PEG-2000 immunotoxin,metoclopramide, 7-thia-8-oxoguanosine, 90:10:5 (w/w);N-solanesyl-N,N′-bis(3,4- DMPC/DMPE-PEG-dimethoxybenzyl)ethylenediamine, N-[4[(4- 2000 fluorphenyl)sulfonyl]phenyl] acetamide, leucovorin, 95:5 (w/w); heparin, heparin sulfate,cimetidine, a radiosensitizer, a DMPC/DSPE-PEG- chemosensitizer, ahypoxic cell cytotoxic agent, 2000 muramyl dipeptide, vitamin A,2′-deoxycoformycin, a 95:5 (w/w) bis-diketopiperazine derivative havingpotentiator activity, dimethyl sulfoxide or mixtures thereof

Examples of malignant diseases for treatment using the present inventioninclude, but are not limited to a cancer of the skin, the GI-tract(esophagus, stomach, small and large intestines), the lungs, the liver,the pancreas, the brain, the breasts, the prostate, the uterine cervixand vagina, head and neck and components of the hematopoietic system(leukemias, lymphomas). Non-limiting examples of non-malignant tissueproliferative disease include gastrointestinal polyp formation, multiplepolyposis and neurofibromatosis. Non-limiting examples of autoimmune oranti-inflammatory include anaphylaxis, arthritis, or irritable bowelsyndrome. Examples of neurodegenerative diseases include, but are notlimited to, fronto-temporal dementia, Alzheimer's disease, Parkinson'sdisease, Huntington's disease, carpal tunnel syndrome and amyotrophiclateral sclerosis (ALS). Non-limiting examples of degenerative diseasesof the soft-tissue that may be treated using the present inventioninclude cataracts, arthritis, neural disease, muscular disease,connective tissue disease, or a combination thereof. For the method oftreatment and medicaments prepared for use in treated the diseases, thecurcumin may be selected from the group consisting of Ar-tumerone,methylcurcumin, demethoxy curcumin, bisdemethoxycurcumin, sodiumcurcuminate, dibenzoylmethane, acetylcurcumin, feruloyl methane,tetrahydrocurcumin,1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione (curcumin1),1,7-bis(piperonyl)-1,6-heptadiene-3,5-dione (piperonyl curcumin)1,7-bis(2-hydroxy naphthyl)-1,6-heptadiene-2,5-dione (2-hydroxylnaphthyl curcumin) and 1,1-bis(phenyl)-1,3,8,10 undecatetraene-5,7-dioneand combinations thereof.

TABLE IX Lipid composition Potentiator Neurodegenerative/InflammatoryDiseases DMPC/Chol/ 0.1-1,000 mg Alzheimer's Disease:acetylcholinesterase inhibitors DMPE-PEG-2000 (e.g., donepezil,galantamine and rivastigmine, which 90:10:5 (w/w); may be provided inoral form and taken once or twice a DMPC/Chol/ day (or less aspotentiators) or even as a transdermal DSPE-PEG-2000 patch); Ginkgobiloba; and N-methyl, D-Aspartate 90:10:5 (w/w); (NMDA) antagonists suchas dextromethorphan, DMPC/DMPE-PEG- dextrorphan, ibogaine, ketamine,nitrous oxide, 2000 phencyclidine, memantine, amantadine or tramadol95:5 (w/w); DMPC/DSPE-PEG- 2000 95:5 (w/w) DMPC/Chol/ 0.1-1,000 mgParkinson's Disease: dopamine-agonists (e.g., DMPE-PEG-2000bromocriptine, pergolide, pramipexole, ropinirole, 90:10:5 (w/w);cabergoline, apomorphine, lisuride), monoamine DMPC/Chol/ oxidase-B(MAO-B) inhibitors (e.g., rasagiline, DSPE-PEG-2000 selegiline) orL-DOPA (or related enzyme inhibitors, 90:10:5 (w/w); e.g., carbidopa,benserazide, tolcapone, entacapone, DMPC/DMPE-PEG- carbidopa/levodopaand benserazide/levodopa). 2000 95:5 (w/w); DMPC/DSPE-PEG- 2000 95:5(w/w) DMPC/Chol/ 0.1-1,000 mg Autoimmune and autoinflammatory diseasesinclude DMPE-PEG-2000 immunosuppressive or anti-inflammatory (e.g.,90:10:5 (w/w); cyclosporine, steroids (e.g., prostaglandins), FK-506,DMPC/Chol/ Non-steroidal anti-inflammatory drugs (NSAIDs)(e.g.,DSPE-PEG-2000 ibuprofen, diclofenac, aspirin, naproxen), and natural90:10:5 (w/w); products (e.g., capsaicin, hyssop, ginger, helenalin,DMPC/DMPE-PEG- willow bark) 2000 95:5 (w/w); DMPC/DSPE-PEG- 2000 95:5(w/w)

Example 20

In another example, the present invention is a method of treating aparasitic infection by contacting the parasite with an effective amountof a curcuminoid:liposome complex effective to treat the parasiticinfection and the curcuminoids are natural or synthetic and/or the useof a medicament with the above characteristics for the treatment ofparasitic infections. Non-limiting examples of parasites that may betreated using the present invention and a medicament directed theretoinclude falciparum hookworm, filiariais, Leishmaniasis, Treponema,Shistosomaisis. The curcuminoid:liposome complex may also include one ormore anti-malarial agents selected from artesiminin, 8-aminoquinoline,amodiaquine, arteether, artemether, artemsinin, artesunate, artesunicacid, artelinic acid, atovoquone, azithromycine, biguanide, chloroquine,chloroquine phosphate, chlorproguanil, cycloguanil, dapsone, desbutylhalofantrine, desipramine, doxycycline, dihydrofolate, reductaseinhibitors, dipyridamole, halofantrine, haloperidol, hydroxychloroquinesulfate, imipramine, mefloquine, penfluridol, phospholipid inhibitors,primaquine, proguanil, pyrimethamine, pyronaridine, quinine, quinidine,quinacrineartemisinin, sulfonamides, sulfones, sulfadoxine, sulfalene,tafenoquine, tetracycline, tetrandine, triazine, salts and derivativesthereof.

TABLE X Lipid composition Potentiator Parasitic Disease DMPC/Chol/0.1-1,000 mg artesiminin, 8-aminoquinoline, amodiaquine, arteether,DMPE-PEG-2000 artemether, artemsinin, artesunate, artesunic acid,90:10:5 (w/w); artelinic acid, atovoquone, azithromycine, biguanide,DMPC/Chol/ chloroquine, chloroquine phosphate, chlorproguanil,DSPE-PEG-2000 cycloguanil, dapsone, desbutyl halofantrine, 90:10:5(w/w); desipramine, doxycycline, dihydrofolate, reductase DMPC/DMPE-PEG-inhibitors, dipyridamole, halofantrine, haloperidol, 2000hydroxychloroquine sulfate, imipramine, mefloquine, 95:5 (w/w);penfluridol, phospholipid inhibitors, primaquine, DMPC/DSPE-PEG-proguanil, pyrimethamine, pyronaridine, quinine, 2000 quinidine,quinacrineartemisinin, sulfonamides, 95:5 (w/w) sulfones, sulfadoxine,sulfalene, tafenoquine, tetracycline, tetrandine, triazine, salts andderivatives thereof.

For the method of treatment and medicaments prepared for use in treatedthe parasitic infections, the curcumin may be selected from the groupconsisting of Ar-tumerone, methylcurcumin, demethoxy curcumin,bisdemethoxycurcumin, sodium curcuminate, dibenzoylmethane,acetylcurcumin, feruloyl methane, tetrahydrocurcumin,1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione (curcumin1),1,7-bis(piperonyl)-1,6-heptadiene-3,5-dione (piperonyl curcumin)1,7-bis(2-hydroxy naphthyl)-1,6-heptadiene-2,5-dione (2-hydroxylnaphthyl curcumin) and 1,1-bis(phenyl)-1,3,8,10 undecatetraene-5,7-dioneand combinations thereof.

Example 21

In operation, the present invention also includes a method of treating anon-human animal comprising: providing the non-human animal with aneffective amount of a curcuminoid:liposome complex effective to loadcurcumin into the liposome, wherein the curcuminoids comprises between 2to 9 weight percent of the total composition effective to treat thenon-human animal and the curcuminoids are natural or synthetic and thecurcuminoid:liposome complex is PEGylated. The curcumin may be selectedfrom the group consisting of Ar-tumerone, methylcurcumin, demethoxycurcumin, bisdemethoxycurcumin, sodium curcuminate, dibenzoylmethane,acetylcurcumin, feruloyl methane, tetrahydrocurcumin,1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione (curcumin1),1,7-bis(piperonyl)-1,6-heptadiene-3,5-dione (piperonyl curcumin)1,7-bis(2-hydroxy naphthyl)-1,6-heptadiene-2,5-dione (2-hydroxylnaphthyl curcumin) and 1,1-bis(phenyl)-1,3,8,10 undecatetraene-5,7-dioneand combinations thereof. In one aspect, the curcuminoid:liposomecomplex comprises sterically-stabilized liposomes. Non-limiting examplesof non-human animal include a horse, a cat, a dog, a hamster, a pig, acow, a goat or a non-domesticated animal.

Example 22

Another example of the present invention is a method of treating a humanwith iron overload or hemochromatosis with a liposomal curcumin orliposomal curcuminoids complex, wherein the complex may be PEGylated ornon-PEGylated and the curcuminoids are natural or synthetic. Theperiodic phlebotomies may be reduced in number, length and/or frequencybased on the improved condition of the patient.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and/or methods and in the steps or in the sequence of stepsof the methods described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents that are chemically or physiologicallyrelated may be substituted for the agents described herein while thesame or similar results would be achieved. All such similar substitutesand modifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

REFERENCES

-   1. Allen and Choun, FEBS Lett., 223:42-46, 1987.-   2. Araújo and Leon, Mem. Inst. Oswaldo Cruz, 96:723, 2001.-   3. Couvreur et al., FEBS Lett., 84:323-326, 1977.-   4. Couvreur et al., U.S. Patent 4:489-555, 1984.-   5. Couvreur, Crit. Rev. Ther. Drug Carrier Syst., 5:1-20, 1988.-   6. Couvreur et al., Pharm. Res., 8:1079-1086, 1991.-   7. Desiderio and Campbell, J. Reticuloendothel. Soc., 34:279-287,    1983.-   8. Düzgünes et al., Antimicrob. Agents Chemother., 32:1404-1411,    1988.-   9. Fattal et al., Antimicrob. Agents Chemother., 33:1540-1543, 1989.-   10. Fattal et. al., J Microencapsul, 8(1):29-36, 1991a.-   11. Fattal et. al., Antimicrob Agents Chemother, 35(4):770-772,    1991b.-   12. Fountain et al., J. Infect Dis., 152-529-535, 1985.-   13. Gabizon and Papahadjopoulos, Proc. Natl. Acad. Sci. USA,    85:6949-6953, 1988.-   14. Grislain et al., Int. J. Pharm., 15:335-345, 1983.-   15. Henry-Michelland et al., Int. J. Pharm., 35:121-127, 1987.-   16. Ireson, C., Cancer Research 61:1058-64, 2001-   17. John et al., J. Exp. Clin. Cancer Res., 21:219-24, 2002.-   18. Lin et al., Cancer Lett. 168:125, 2001.-   19. Mastrobattista et al., Advanced Drug Delivery Reviews,    40:103-180.-   20. Mosman T, J. Immunol. Methods, 65:55, 1983.-   21. Poste, Biol. Cell., 47:19-39, 1983.-   22. Tulkens, In P. Buri and R. Gumma (eds.), aims, Potentialities    and Problems in Drug Targeting, Elsevier, Amsterdam, 1985, pp.    179-194.-   23. Woodle et al., Pharmaceutical Research 9, 260-265 (1992)

What is claimed is:
 1. A method of treating a skin, pancreatic, orbreast cancer cell comprising: identifying a patient in need oftreatment for the skin, pancreatic, or breast cancer; and providing thepatient in need thereof with an effective amount of acurcuminoid:PEGylated-liposome, wherein the liposome comprises a ratioof liposome to PEG comprises between 2 to 9 weight percent of the totalcomposition, wherein the PEGylated-liposome is selected from at leastone of DMPC/Chol/DMPE-PEG-2000 liposome at a ratio of between 90:10:2(w/w) to 90:10:9 (w/w); a DMPC/Chol/DSPE-PEG-2000 liposome at a ratio ofbetween 90:10:2 (w/w) to 90:10:9 (w/w); a DMPC/DMPE-PEG-2000 liposome ata ratio of between 90:10:2 (w/w) to 90:10:9 (w/w); or aDMPC/DSPE-PEG-2000 liposome at a ratio of between 90:10:2 (w/w) to90:10:9 (w/w), and the curcuminoid is at between 2 to 9 weight percent.2. The method of claim 1, wherein the curcuminoid is administered in adose of from about 0.01 mg/kg of the individual's body weight to about500 mg/kg of the individual's body weight.
 3. The method of claim 1,wherein the curcuminoid is selected from the group consisting ofAr-tumerone, methylcurcumin, demethoxy curcumin, bisdemethoxycurcumin,sodium curcuminate, dibenzoylmethane, acetylcurcumin, feruloyl methane,tetrahydrocurcumin,1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione (curcumin),1,7-bis(piperonyl)-1,6-heptadiene-3,5-dione (piperonyl curcumin)1,7-bis(2-hydroxy naphthyl)-1,6-heptadiene-2,5-dione (2-hydroxylnaphthyl curcumin) and 1,1-bis(phenyl)-1,3,8,10undecatetraene-5,7-dione.